NZ627549B2 - A combination therapy for a stable and long term engraftment using specific protocols for t/b cell depletion - Google Patents

Abstract

Disclosed is the use of a dose of T cell depleted immature hematopoietic cells which are obtained by a specific process as defined herein 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. or immature hematopoietic cell transplantation.

Description

A COMBINATION THERAPY FOR A STABLE AND LONG TERM ENGRAFTMENT USING SPECIFIC PROTOCOLS FOR T/B CELL DEPLETION RELATED APPLICATION/S US. Provisional Patent Application No. 61/578,917 filed on December 22, 2011, which is hereby incorporated by reference as if fully set forth herein.

FIELD AND OUND 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 use of full—haplotype mismatched haploidentical donors as an alternative source for poietic stem cell transplantation (HSCT) is highly attractive since virtually all patients have a readily ble haploidentical family member that can serve as an HSCT donor. Early attempts 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 resistant 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 ted sfully even when using fully mismatched murine strain ations [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 tion may be used to overcome genetic barriers, enabling satisfactory survival rates following purified haploidentical HSCT er Y and Martelli MF. Immunol Today. (1995) 16:437-440 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 depleted transplants, is the slow recovery rate of the recipient’s immune system.

This is attributed to extensive 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 ents. Thus, in adult recipients of a haploidentical CD34+ stem cell graft, a significant rate of lant related mortality (TRM) is caused by opportunistic infections.

Several approaches are being developed to address this challenge. This es novel modalities to improve thymic on, post-transplant adoptive transfer of anti- viral specific T cells, transfer of partially polyclonal host-non-reactive epleted T cells or transfer of fully polyclonal T cells transfected with inducible suicide genes. An alternative and additional approach to preserve host immunity is the use of reduced intensity conditioning (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 ioning agents. Haploidentical transplantation under RIC is even more ate due to the substantial immunological barrier presented by the ing host T cells. Recent attempts to me 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 approach for applying haploidentical transplantation under RIC uses CD3/CD19 depleted , 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 trated in rodents that a short course of high- dose cyclophosphamide (CY) soon after bone marrow transplant (BMT) targeted activated donor or host alloreactive T cells [Owens AH Jr and GW. S. Transplantation. (1971) 11:378~382]. Cyclophosphamide was observed to be non—toxic to hematopoietic stem cells because of their high expression of the detoxifying enzyme aldehyde dehydrogenase, and Slavin et a1. further demonstrated that administration of high dose hosphamide can reduce GVHD and graft rejection in mice, t adverse effects on stem cell engraftment [Brodsky RA and R]. J. Lancet. (2005) 365:1647— 1656]. Clinical trials by the John Hopkins and Fred nson Cancer Research Center groups, ted a non—myeloablative protocol of cyclophosphamide, fludarabine and 2Gy TBI, and post—transplant GVHD prophylaxis with cyclophosphamide (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: journal of the American Society for Blood and Marrow Transpiantation. (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., Pediatric Reports (2011) 3:43-47].

Additional approaches for achieving stable engraftment of allogeneic hematopoietic stem cells have been attempted, some are described in US. Patent Application No. 20110110909, US. Patent Application No. 20050118142, US. Patent Application No. 20070098693, US. Patent No. 692, US. Patent No. 5,514,364, US. Patent No. 6,217,867, US. Patent No. 5,635,156, US. Patent Application No. 20060140912, US. Patent Application No. 20040005300, US. Patent Application No. 20070141027, US. Patent Application No. 20030017152, US. Patent Application No. 20030165475 and US. Patent Application No. 20010009663.

SUMMARY OF THE INVENTION In a first aspect, the present invention provides the use of a dose of T cell depleted re hematopoietic 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 wherein said T cell depleted immature hematopoietic cells are obtained in Vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells comprising the re poietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said re hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) g said matrix to remove unbound cells; and (iv) removing said magnetic field to elute bound cells from said matrix; 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 hosphamide is for administration to the subject ing transplantation of a cell or tissue graft, in the manufacture of a ment for treating a subject in need of a non-syngeneic cell or tissue graft, n said subject has a malignant disease. (followed by page 3a) In another 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 ses at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained in Vitro by separating said T cells from said re hematopoietic cells, by a method sing: (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that specifically binds to a surface , n said antibody is labeled with a ically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) washing said matrix to remove d cells; and (iv) removing said magnetic field to elute bound cells from said matrix; and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises -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 ngeneic cell or tissue graft, wherein said subject has a non—malignant disease.

In still a further aspect of the invention, there is provided the use of a dose of T cell ed immature hematopoietic cells, wherein said T cell depleted 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 kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix h a magnetic field; (iii) washing said matrix to remove unbound cells; and (iv) removing said ic field to elute bound cells from said matrix; and a therapeutically effective (followed by page 3b) amount of hosphamide, wherein said therapeutically effective amount comprises -200 mg per kilogram body weight of the subject, and wherein said cyclophosphamide is for stration to the t following transplantation of immature poietic cells, in the manufacture of a medicament for treating a subject in need of an immature hematopoietic cell transplantation, 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, 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 wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that cally binds to a surface marker, wherein said antibody is d with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically sive agent in a matrix through a magnetic field; (iii) washing said matrix to remove unbound cells; and (iv) ng said magnetic field to elute bound cells from said matrix; and a therapeutically effective amount of hosphamide, wherein said therapeutically effective amount ses —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, wherein said subject has a non-malignant disease.

In a fiirther aspect of the invention there is provided the use of a reduced intensity conditioning protocol, wherein said reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent; a dose of T cell depleted re 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 ses at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted re hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (followed by page 3c) (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that specifically binds to a' surface marker, wherein said antibody is labeled with a ically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) washing said matrix to remove d cells; and (iv) removing said magnetic field to elute bound cells from said matrix; 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 ng a subject in need of an immature hematopoietic cell transplantation, n said subject has a malignant e.

In another aspect of the invention there is provided the use of a reduced intensity conditioning protocol, wherein said reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent; a dose of T cell ed 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 n said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method sing: (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) washing said matrix to remove d cells; and (iv) removing said magnetic field to elute bound cells from said ; and a therapeutically effective amount of cyclophosphamide, n said therapeutically effective amount ses 25-200 mg per kilogram body weight of the subject, and n said cyclophosphamide is for stration to the subject following transplantation of immature hematopoietic cells, in the manufacture of a (followed by page 3d) medicament for treating a t in need of an immature hematopoietic cell transplantation, wherein said subject has a non-malignant disease.

In a further aspect of the ion, there is provided the use of a dose of T cell depleted immature hematopoietic cells from a non-syngeneio donor, wherein said T cell ed 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 wherein said T cell depleted immature hematopoietic cells are obtained in Vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a tion of cells comprising the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) g said matrix to remove unbound cells; and (iv) removing said magnetic field to elute bound cells from said ; and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg per am 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 non—syngeneic cell or tissue graft, wherein said t has a malignant disease.

In a still further aspect of the invention there is provided the use of a dose of T cell depleted immature poietic cells from a non-syngeneic donor, n said T cell depleted immature 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 wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells comprising the re hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells (followed by page 3e) specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) washing said matrix to remove d cells; and (iv) removing said magnetic field to elute bound cells from said matrix; 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 ng donor specific tolerance in a subject in need of a ngeneic cell or tissue graft, wherein said subject has a non—malignant e.

In another 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 poietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose ses at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein separating said T cells from said immature hematopoietic cells is effected in Vitro based on a product ed by said T cells; and a therapeutically effective amount of cyclophosphamide, wherein said eutically effective amount comprises 25—200 mg per kilogram body weight of the subject, and wherein said cyclophosphamide 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 malignant disease.

In a further aspect of the ion there is provided the use of a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted 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 kilogram body weight of the subject, and wherein separating said T cells from said immature hematopoietic cells is effected in Vitro based on a product secreted by said T cells; and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 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 (followed by page 3f) subject in need of a non—syngeneic cell or tissue graft, wherein said subject has a non— malignant disease.

In another aspect of the invention there is ed the use of a dose of T cell ed immature hematopoietic cells, wherein 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 subject, and wherein said T cell ed immature hematopoietic cells are obtained in Vitro by separating said T cells from said immature hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of CD2, CD3, CD4, CD8, TCR d/[i and TCR 7/5; and a therapeutically effective amount of cyclophosphamide, 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 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 a further 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 wherein said T cell depleted immature hematopoietic cells are obtained in Vitro by separating said T cells from said immature poietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of CD2, CD3, CD4, CD8, TCR (it/B and TCR y/5; 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 a cell or tissue graft, in the manufacture of a medicament for ng a subject in need of a non- syngeneic cell or tissue graft, wherein said subject has a non—malignant disease.

In another aspect of the ion there is provided use of 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 (followed by page 3g) said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the t, and wherein separating said T cells from said immature poietic cells is effected in vitro based on a t secreted by said T cells; and a therapeutically effective amount of cyclophosphamide, n 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 cture of a medicament for treating a conditioned subject in need of an immature hematopoietic cell transplantation, wherein said subject has a malignant disease.

In a still further aspect of the invention there is provided the use of a dose of T cell depleted immature hematopoietic cells, wherein 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 subject, and wherein separating said T cells from said immature hematopoietic cells is effected in vitro based on a product secreted by said T cells; 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 cyclophosphamide is for administration to the t following lantation of immature hematopoietic cells, in the manufacture of a medicament for treating a conditioned subject in need of an immature hematopoietic cell transplantation, wherein said subject has a non-malignant disease.

In another 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 ses at least about 5 X 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said re hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of CD2, CD3, CD4, CD8, TCR (1/6 and TCR 7/6; and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25—200 mg per kilogram body weight of the t, and wherein said (followed by page 3h) cyclophosphamide is for administration to the subject following lantation of immature hematopoietic cells, in the manufacture of a medicament for treating a conditioned subject in need of an immature hematopoietic cell transplantation, wherein said subject has a malignant disease.

In a further aspect of the invention there is provided the use of a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted re hematopoietic cells comprise less than 5 X 105 CD3+ cells per kilogram body weight of a subject, and n said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell ed immature poietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells based on an expression of at least one e marker of T cells selected from the group consisting of CD2, CD3, CD4, CD8, TCR (X/B and TCR 7/8; 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 immature hematopoietic cell transplantation, wherein said subject has a non-malignant disease.

In a still further aspect of the invention there is ed 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 re 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 n said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein separating said T cells from said immature hematopoietic cells is effected in vitro based on a t secreted by said T cells; and a therapeutically effective amount of cyclophosphamide, 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 subject (followed by page 3i) in need of an immature hematopoietic cell transplantation, wherein said subject has a malignant disease.

In another aspect of the invention there is ed use of a reduced intensity 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 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 wherein separating said T cells from said immature hematopoietic cells is effected in vitro based on a product secreted by said T cells; 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 subject in need of an immature hematopoietic cell lantation, n said subject has a lignant disease.

In a still further aspect of the invention there is provided use of a reduced intensity conditioning protocol, wherein said reduced intensity conditioning ses a total body irradiation (TBI) and a chemotherapeutic agent; a dose of T cell depleted immature hematopoietic cells, wherein said T cell ed re hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and n said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted re hematopoietic cells are ed in vitro by separating said T cells from said immature hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of CD2, CD3, CD4, CD8, TCR Ot/B and TCR y/S; 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 t in need of an re hematopoietic cell transplantation, wherein said subject has a malignant disease. (followed by page 3]) In yet another aspect of the present invention there is provided the 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 hematopoietic cells, wherein said T cell depleted immature poietic cells se less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and n said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of CD2, CD3, CD4, CD8, TCR a/B and TCR y/S; and a therapeutically effective amount of cyclophosphamide, wherein said eutically 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 ment for treating a subject in need of an immature hematopoietic cell transplantation, wherein said t 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, wherein said T cell ed immature hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose ses at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and n separating said T cells from said immature hematopoietic cells is effected in vitro based on a product secreted by said T cells; 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 inducing donor specific tolerance in a subject in need of a non-syngeneic 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 ed immature hematopoietic cells from a non-syngeneic donor, wherein said T cell depleted 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+ (followed by page 3k) cells per kilogram body weight of the subject, and wherein separating said T cells from said immature hematopoietic cells is effected in vitro based on a product secreted by said T cells; 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 lantation of a cell or tissue graft, in the manufacture of a medicament for inducing donor specific tolerance in a subject in need of a non—syngeneic cell or tissue graft, wherein said subject has a non-malignant disease.

In a 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, wherein said T cell depleted immature poietic cells comprise less than 5 x 105 CD3)r cells per kilogram body weight of a subject, and n said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of CD2, CD3, CD4, CD8, TCR (1/6 and TCR 31/8; 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 lantation of a cell or tissue graft, in the manufacture of a medicament for inducing donor specific tolerance in a subject in need of a non-syngeneic cell or tissue graft, n said subject has a malignant disease.

In yet a further 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 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 wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said re hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group ting of CD2, CD3, CD4, CD8, TCR (“l/B and TCR 7/6; and a therapeutically effective amount of cyclophosphamide, n said eutically (followed by page 3|) 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 inducing donor specific nce in a subject in need of a non-syngeneic cell or tissue graft, wherein said subject has a non-malignant disease.

According to an aspect of some embodiments of the present ion there is provided a method of treating a subject in need of a nomsyngeneic cell or tissue graft, the method comprising: (a) lanting into a 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 comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein the T cell depleted immature hematopoietic cells are obtained by separating the T cells from the immature hematopoietic cells, by a method comprising: (i) adding an antibody to the immature hematopoietic cells that specifically binds to a surface marker, wherein the antibody is labeled with a ically responsive agent; (ii) immobilizing the immature poietic cells specifically bound to the antibody labeled with the magnetically responsive agent in a matrix through a magnetic field; (iii) washing the matrix to remove unbound cells; and (iv) removing the magnetic field to elute bound cells from the matrix; and subsequently (b) stering 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.

According to an aspect of some embodiments of the present invention there is provided a method of ng a subject in need of an immature hematopoietic cell [FOLLOWED BY PAGE 4] transplantation, the method comprising: (a) transplanting into a conditioned subject a dose of T cell depleted immature hematopoietic cells, wherein the T cell depleted immature poietic 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 wherein the T cell depleted immature hematopoietic cells are obtained by separating the T cells from the immature hematopoietic cells, by a method comprising: (i) adding an dy to the immature hematopoietic cells that specifically binds to a e marker, wherein the antibody is labeled with a magnetically responsive agent; (ii) immobilizing the immature hematopoietic cells specifically bound to the antibody labeled with the magnetically responsive agent in a matrix through a magnetic field; (iii) g the matrix to remove unbound cells; and (iv) removing the magnetic field to elute bound cells from the matrix; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, n the eutically 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 treating a t in need of an immature hematopoietic cell transplantation, the method comprising: (a) conditioning a subject under a d ity ioning ol, wherein the reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent; (b) transplanting into the subject a dose of T cell depleted immature hematopoietic cells, wherein the T cell depleted immature poietic 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 wherein the T cell depleted immature hematopoietic cells are obtained by separating the T cells from the immature hematopoietic cells, by a method comprising: (i) adding an antibody to the immature poietic cells that specifically binds to a surface marker, wherein the antibody is labeled with a magnetically responsive agent; (ii) immobilizing the immature hematopoietic cells specifically bound to the antibody labeled with the magnetically responsive agent in a matrix through a magnetic field; (iii) washing the matrix to remove unbound cells; and (iv) removing the magnetic field to elute bound cells from the matrix; and subsequently (c) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the eutically effective amount ses 25-200 mg per kilogram body weight, thereby treating the subject.

According to an aspect of some ments of the present invention there is provided a method of inducing donor specific tolerance in 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 depleted immature hematopoietic cells obtained from a non—syngeneic donor, wherein the T cell depleted immature poietic 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 wherein the T cell depleted immature hematopoietic cells are obtained by separating the T cells from the immature hematopoietic cells, by a method comprising: (i) adding an antibody to the immature hematopoietic cells that specifically binds to a surface , n the antibody is labeled with a magnetically responsive agent; (ii) immobilizing the immature hematopoietic cells specifically bound to the antibody labeled with the magnetically sive agent in a matrix through a magnetic field; (iii) washing the matrix to remove unbound cells; and (iv) removing the ic field to elute bound cells from the matrix; 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 t.

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 subject a dose of T cell depleted immature 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 comprises at least about 5 X 106 CD34+ cells per am body weight of the subject, and wherein the ting the T cells from the immature hematopoietic cells is effected based on a product ed by the T cells; and uently (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 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 subject a dose of T cell depleted immature hematopoietic cells, wherein the T cell depleted immature hematopoietic cells se 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 wherein the T cell depleted immature hematopoietic cells are obtained by separating the T cells from the immature hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of 4-1BB, FoxP3, CD154, CD4, CD8, CD25, GITR CD137, latent TGF—beta (LAP), GARP 2) and /b; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the eutically effective amount comprises 25—200 mg per kilogram body weight, thereby treating the subject.

According to an aspect of some embodiments of the t 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 t a dose of T cell depleted immature hematopoietic cells, wherein the T cell depleted re 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 wherein the separating the T cells from the re hematopoietic cells is effected based on a product secreted by the T cells; and subsequently (b) administering to the subject a eutically 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 some embodiments of the present invention there is ed a method of treating a t in need of an immature hematopoietic cell lantation, the method sing: (a) transplanting into a conditioned subject a dose of T cell depleted immature 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 comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein the T cell depleted immature 2012/050541 hematopoietic cells are obtained by separating the T cells from the immature poietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of 4-lBB, FoxP3, CD154, CD4, CD8, CD25, GITR CD137, latent TGF—beta (LAP), GARP 2) and CD12la/b; and subsequently (b) stering to the subject a therapeutically ive amount of hosphamide, wherein the therapeutically effective amount ses 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 treating a subject in need of an re hematopoietic cell transplantation, the method comprising: (a) conditioning a subject under a reduced intensity ioning protocol, wherein the d ity conditioning comprises a total body ation (TBI) and a chemotherapeutic agent; (b) transplanting into the subject a dose of T cell depleted immature hematopoietic cells, wherein the T cell depleted immamre 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 am body weight of the subject, and wherein the separating the T cells from the immature hematopoietic cells is effected based on a product secreted by the T cells; and subsequently (c) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount ses 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 treating a subject in need of an immature hematopoietic cell transplantation, the method comprising: (a) conditioning a subject under a reduced intensity conditioning protocol, wherein the reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent; (b) transplanting into the subject a dose of T cell depleted immature 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 comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein the T cell depleted immature hematopoietic cells are obtained by separating the T cells from the immature hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of 4—lBB, FoxP3, CD154, CD4, CD8, CD25, GITR CD137, latent TGF-beta (LAP), GARP 2) and CDlZla/b; 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.

According to an aspect of some ments of the present invention there is provided a method of inducing donor specific tolerance in a t in need of a non— syngeneic 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 wherein the separating the T cells from the immature hematopoietic cells is effected based on a product secreted by the T cells; and subsequently (b) administering to the t a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount ses 25-200 mg per kilogram body weight, thereby inducing donor specific tolerance in the subject.

According to an aspect of some ments of the present invention there is provided a method of ng donor c tolerance in a subject in need of a nonsyngeneic cell or tissue graft, the method comprising: (a) transplanting into a subject a dose of T cell depleted immature 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 wherein the T cell ed immature hematopoietic cells are ed by separating the T cells from the immature hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of 4—1BB, FoxP3, CD154, CD4, CD8, CD25, GITR CD137, latent TGF—beta (LAP), GARP (LRRC32) and CD121a/b; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount ses 25—200 mg per am 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 r comprises conditioning the subject with in-vivo T cell debulking prior to step (a).

According to some embodiments of the ion, the dose of the T cell depleted immature hematopoietic cells ses 5 ~ 40 X 106 CD34+ cells per kilogram body weight of the subject.

According to some embodiments of the ion, 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. ing to some ments 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, purified CD34+ cells attained by positive selection from bone marrow and/or from G—CSF mobilized peripheral 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+ TCRa/B' cells per kilogram body weight of the subject.

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

According to some embodiments of the invention, when the e marker is a T cell surface marker, the d cells comprise T cell depleted immature hematopoietic cells.

According to some embodiments of the invention, the T cell e marker is selected from the group consisting of CD2, CD3, CD4, CD8 and TCRn/B.

According to some embodiments of the invention, when the surface marker is an immature poietic cell surface marker, the bound cells comprise T cell depleted immature hematopoietic cells.

According to some embodiments of the invention, the immature hematopoietic cell surface marker is selected from the group consisting of CD34, CD33 and CD131.

PCT/ILZOIZ/050541 According to some embodiments of the invention, the method further comprises separating B cells from the T cell depleted immature hematopoietic cells by the use of an antibody that specifically binds to a B cell surface marker.

According to some embodiments of the invention, the B cell surface marker is selected from the group ting of CD19 and CD20.

According to some embodiments of the invention, the matrix is a ferromagnetic matrix.

According to some embodiments of the invention, the matrix comprises spheres of magnetically susceptible or ferromagnetic material. ing to some embodiments of the invention, the magnetically responsive agent comprises a superparamagnetic particle.

According to some embodiments of the invention, the superparamagnetic le is conjugated to the antibody in combination with an anti~immunoglobulin, an avidin and or anti—hapten—specific microbead. ing to some embodiments of the invention, the ting the T cells from the immature hematopoietic cells is effected using a high gradient magnetic separation (HGMS).

According to some embodiments of the invention, the separating the T cells from the immature hematopoietic cells is effected using a separation column. ing to some embodiments of the invention, the antibody is selected from the group consisting of an anti—CD8 dy, an anti-CD4 antibody, an D3 dy, an anti—CD2 antibody, an anti~TCRa/B antibody, an anti—CD19 antibody, and an anti—CD20 antibody, an anti—CD21 antibody an anti—CD34 antibody, an anti—CD33 antibody and an anti—CD131 antibody. ing 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 neic with t to the subject.

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

W0 2013l093919 According to some embodiments of the invention, the subject is a human subject.

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

According to some embodiments of the invention, the antibodies comprise at least one of an anti—CD8 antibody, an anti—CD4 antibody, an anti—thymocyte globulin (ATG) antibody, an anti—CD52 antibody and an anti~CD3 (OKT3) antibody.

According to some embodiments of the invention, the reduced intensity conditioning comprises a non—myeloablative 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 therapy.

According to some embodiments of the invention, the TBI comprises a single or fractionated ation dose within the range selected from the group consisting of 1-75 Gy and 1—3.5 Gy.

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

According to some embodiments of the invention, the antibody comprises at least one of an D52 antibody, an anti—thymocyte globulin (ATG) antibody and anti- CD3 (OKT3) antibody.

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

According to some embodiments of the ion, the cyclophosphamide is administered in a single dose or 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 ion, each of the two doses is administered on days 3 and 4 following step (a).

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

WO 93919 According to some embodiments of the invention, the subject has a non- malignant disease.

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

According to some ments of the invention, the cell or tissue graft is selected from the group consisting of a liver, a pancreas, a spleen, a , 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 lanted into the subject prior to, itantly with or following the transplantng the dose of T cell depleted immature hematopoietic cells into the subject.

According 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 immature hematopoietic cells are obtained from the same donor.

According to some embodiments of the invention, the separating is effected using an antibody.

According to some embodiments of the invention, the antibody is coupled to a fluorescent dye, a hapten or a magnetic particle.

According to some embodiments of the ion, the separating is performed using flow—cytometry or magnetic cell sorting. ing to some embodiments of the invention, the magnetic cell sorting comprises MACSTM.

According to some ments of the invention, the separating the T cells from the immature hematopoietic cells based on the product secreted by the T cells comprises separating T cells labeled with a secretion product, wherein the T cells have been coupled to a capture moiety that specifically binds a product secreted by the T cells and n the T cells have been cultured under conditions wherein the product is secreted and bound to the capture moiety, thereby producing T cells labeled with the secretion product, wherein the T cells are not lysed by the method and wherein the secretion product is labeled with a label moiety.

According to some embodiments of the invention, the method further comprises separating B cells from the T cell depleted re hematopoietic cells based on a product secreted by the B cells comprising separating B cells d with a secretion product, n the B cells have been coupled to a capture moiety that specifically binds a product secreted by the B cells and wherein the B cells have been cultured under conditions n the product is secreted and bound to the capture moiety, thereby producing B cells labeled with the secretion product, wherein the B cells are not lysed by the method and wherein the secretion t is labeled with a label . ing to some embodiments of the invention, the secretion product is a cytokine, antibody or hormone.

According to some embodiments of the invention, the secretion product is selected from the group consisting of IFN—y, lLl, 1L2, 1L4, ILlO, ILlZ, TGF—B, TNF, GM-CSF and SCF.

According to some embodiments of the invention, the capture moiety is coupled to the T cells or B cells through an anchoring moiety.

According to some embodiments of the invention, the capture moiety is an dy or an antigen—binding fragment thereof.

According to some embodiments of the invention, the antibody is bispecific.

According to some embodiments of the invention, the antibody is against a T cell or a B cell surface marker.

According to some embodiments of the invention, the label moiety is an antibody specific for the secretion product.

According to some embodiments of the invention, the label moiety is fluorochromated, magnetizable or comprises magnetic les.

According to some embodiments of the invention, the antibody is selected from the group consisting of an D8 antibody, an anti~CD4 antibody, an anti~CD3 antibody, an anti-CD2 antibody, an anti~TCROt/B antibody, an anti—CD19 antibody, an anti-CD20 antibody, an anti—CD21 antibody an anti—CD34 antibody, an anti—CD33 antibody and an anti-CD131 antibody.

It will be appreciated that the present teachings can be used with other tolerance inducing protocols such as described in PCT publication Nos. , WO 2007/023491 and WO 49935, which are herein incorporated by nce in their entirety.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ry skill in the art to which the ion pertains. Although methods and materials similar or lent to those described herein can be used in the practice or g of embodiments of the invention, exemplary methods and/or als are described below. In case of t, the patent specification, including definitions, will control. In addition, the maten'als, methods, and es are illustrative only and are not intended to be arily 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 , it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how ments of the invention may be practiced.

In the drawings: FIGS. 1A-B are graphs illustrating durable engraftment of mismatched donor bone man‘ow (BM) following transplantation 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 irradiation (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 () and 95 days () post transplant.

FIGS. 2A-C are dot plot graphs illustrating a typical FACS chimerism analysis. depicts that mixed chimerism was achieved in recipients that were transplanted with ‘mega dose’ (25 x 106) rigorously T cell depleted BM and were d with high dose CY. In contrast, recipient mice that received only the conditioning protocol () or which were inoculated with only 5 x 106 BM cells and CY did not exhibit donor type chimerism (). is a graph illustrating durable 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 ed BM and CY did not exhibit mixed ism.

FIGS. 4A—B illustrate transplantation of donor type or 3rd party skin grafts in chimeric mice. is a graft illustrating acceptance (marked by “+”) or rejection (marked by “—“) of donor type (Balb/c) or 3rd party (C57BL/6) skin grafts in recipients 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. 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 chimerism 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 hosphamide (CY) on donor type chimerism in recipients of ‘mega dose’ (25 x 106) T ed BM and 2 Gy TBI. is a graph illustrating tment of mismatched donor BM d by combining ‘mega dose’ CD8’r T cell depleted BM 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 chimeiism achieved when combing ‘mega dose’ T cell depleted BM cells with post transplant CY.

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

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

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its ation to the details set forth in the following description or exemplified by the Examples. The ion is capable of other embodiments or of being practiced or carried out in various 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 allogeneic hematopoietic stem cell transplantation (HSCT) has been limited by the lack of available HLA—matched donors within the family or in the international registries of unrelated eer 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 e (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 present invention to ce, the present inventors have red that a successful tment 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 immunomyeloablative conditioning regimen. The present inventors have further shown that such a transplantation ure leads to a long and stable ism and that nce 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 ched donor bone marrow (see Figures lA—B and 2A—C). Durable mixed ism was exhibited for prolonged periods of time after transplantation (180 and 225 days post lant 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 resulted in tolerance induction, 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 depleted immature hematopoietic cells, wherein the T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD35r T cells per am 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) stering to the subject a therapeutically effective amount of hosphamide, wherein the therapeutically effective amount comprises —200 mg per kilogram body , thereby treating the subject.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating al or aesthetical symptoms of a condition or ntially preventing the appearance of clinical or aesthetical symptoms of a condition.

As used herein, 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 physical, morphological or physiological abnormality which is amenable to ent via cell or tissue transplantation.

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

According to one embodiment the subject has a malignant disease.

According to one embodiment the malignant e is a hematopoietic cancer.

Exemplary hematopoietic cancers include, but are not limited to, acute lymphoblastic leukemia (ALL), T~cell acute lymphocytic leukemia ), acute myelocytic leukemia (AML), acute nonlymphoblastic leukemia (ANLL), chronic lymphocytic leukemia (CLL), chronic myelocytic ia (CML), T—cell prolymphocytic ia, B—cell prolymphocytic ia, Juvenile myelomonocytic leukemia, n'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, Diffuse large B-cell lymphoma (DLBCL), B-cell chronic lymphocytic leukemia (B-CLL)/chronic lymphoid leukemia (CLL), Chronic lymphocytic ia/small lymphocytic lymphoma, Extranodal marginal zone B—cell lymphomas - mucosa~associated lymphoid tissue lymphomas, ular lymphoma, Mantle cell lymphoma, Nodal marginal zone B—cell lymphoma, Burkitt lymphoma, Hairy cell ia, Primary central s system lymphoma, Splenic marginal zone B—cell lymphoma, Lymphoplasmocytic lymphoma, Primary mediastinal B—cell ma, precursor T—cell leukemia/lymphoma, MALT lymphoma, Mycosis fungoides and multiple a. ing to one embodiment the poietic cancer comprises a leukemia or a lymphoma.

According to one embodiment the t 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 include, 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 leukodystrophy, thalassemia, congenital or genetically—determined hematopoietic abnormality, adenosine deaminase (ADA), lupus, mune hepatitis, celiac disease, type I diabetes mellitus, Grave's disease, Guillain—Barr syndrome, Myasthenia gravis, toid arthritis, scleroderma and psoriasis.

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

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

According to one ment, the cell or tissue graft ses immature poietic cells.

According to one embodiment, the method is effected using a cell or tissue, which is ngeneic with the subject.

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

As used herein, the term “allogeneic” refers to a cell or tissue which is derived from a donor who is of the same species 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 identical or HLA non—identical (i.e. displaying one or more disparate HLA determinants) with t to the t.

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. lly, outbred mammals of different species are neic with each other.

The present invention envisages that neic 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 domestica), canines (e. g., Canis domestica), rodents (e.g., mouse, rat, rabbit, guinea pig, gerbil, hamster) or primates (e.g., chimpanzee, rhesus monkey, macaque monkey, marmoset). 42012/050541 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. rly, human-derived cells or tissues are preferably obtained from substantially pathogen—free sources.

According to an embodiment of the present ion, 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 cadaver donor. Moreover, ing 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 alt.

Furthermore, any method known in the an 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 sing immature hematopoietic cells (including 6.g. CD34+), are transplanted into a subject.

According to one embodiment, the T cell depleted immature poietic cells are non-syngeneic (e.g. allogeneic or xenogeneic) with the subject.

According 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 hematopoietic tissue or cell preparation sing precursor hematopoietic cells. Such tissue/cell preparation includes or is d from a biological sample, for example, bone marrow, mobilized peripheral blood (e. g. mobilization of CD34 cells to e their tration), cord blood (e.g. 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 expansion.

According to one embodiment, the immature hematopoietic cells compiise T cell depleted immature hematopoietic cells.

As used herein the phrase “T cell ed immature hematopoietic cells” refers to a population of hematopoietic cells which are depleted of T lymphocytes. The T cell depleted immature poietic cells, may include e.g. CD34+, CD33+ and/or CD56+ cells. The T cell depleted immature hematopoietic cells may be depleted of CD3+ cells, CD2+ cells, CD8+ cells, CD4+ cells, a/B T cells and/or 7/8 T cells.

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

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

According to an ment, the T cell depleted immature hematopoietic cells comprise less than 50 X 105 CD3+ T cells, 40 X 105 CD3+ T cells, 30 x 105 CD3+ T cells, 20 x lo5 CD3+ T cells, 15 x 105 CD3+ T cells, 10 x lo5 CD3+ T cells, 9 x 105 CD3“ T cells, 8 x 105 CD3+ T cells, 7 x lo5 CD3+ T cells, 6 x 105 CD3+ T cells, 5 x 105 CD3+ T cells, 4 x 105 CD3“ T cells, 3 x lo5 CD3+ T cells, 2 x lo5 CD3+ T cells or 1 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 5 X 105 CD3+ T cells per kilogram body weight of the t.

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

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

According to an embodiment, the T cell depleted re hematopoietic cells comprise less than 1 X 104— 4 X 105 CD8+ cells per kilogram body weight of the t.

According to an embodiment, the T cell ed immature hematopoietic cells comprise less than 50 x 105 CD8+ cells, 25 X 105 CD8+ cells, 15 X 105 CD8+ cells, 10 X lo5 CD8+ cells, 9 x lo5 CD8+ cells, 8 x 105 CD8+ cells, 7 x 105 CD8+ cells, 6 x 105 CD8+ cells, 5 x 105 CD8+ cells, 4 x lo5 CD8+ cells, 3 x 105 CD8+ cells, 2 x 105 CD8+ cells, 1 x lo5 CD8+ cells, 9 x 104 CD8+ cells, 8 x lo4 CD8+ cells, 7 x 104 CD8+ cells, 6 x 104 CD8+ cells, 5 x 104 CD8+ cells, 4 x lo4 CD8+ cells, 3 x lo4 CD8+ cells, 2 x lo4 CD8+ cells or 1 x 104 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 CD8Jr cells per kilogram body weight of the subject.

WO 2013093919 2012/050541 According to a specific embodiment the T cell ed 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+ TCRu/B— cells per kilogram body weight of the subject.

According to an embodiment, the T cell depleted immature poietic cells se less than 1 x lo6 CD8+ TCRa/B‘ cells, 0.5 x lo6 CD8+ TCRc/B‘ cells, 1 x 105 CD8+ TCRa/B' cells, 0.5 x lo5 CD8+ TCRtx/B' cells, 1 x lo4 CD8+ TCRoz/B' cells, 0.5 x lo4 CD8+ TCRa/B' cells, 1 x 103 CD8+ TCRu/B' cells or 0.5 x 103 CD8+ TCRa/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 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+ B‘ cells but more than 10 CD8+ TCROl/B~ cells.

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

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

According to an embodiment, the immature 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 105 B cells, 8 x 105 B cells, 7 x 105 B cells, 6 x 105 B cells, 5 x 105 B cells, 4 x 105 B cells, 3 x 105 B cells, 2 x 105 B cells or 1 x 105 B cells per am body weight of the subject.

According to a specific embodiment, the immature hematopoietic cells comprise less than 4 x 105 B cells per kilogram body weight of the subject. According to a specific embodiment the immature poietic 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 c antibodies or by affinity based purification such as by the use of ic-activated cell sorting (MACSTM) available W0 20131093919 from Miltenyi Biotec (depicted in further detail below), FACS sorter and/or e ELISA labeling.

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

For example, 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 tion techniques known in the art may be used in conjunction with both positive and negative separation techniques that rely on the physical properties of the cells rather than antibody affinity, ing but not limited to elutriation and density gradient centrifugation.

Other methods for cell sorting include, for e, panning and tion using ty techniques, including those techniques using solid supports such as plates, beads and columns. Thus, biological samples may be separated 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, U.S.A.), Immuncon (Philadelphia, USA), Immunotec (Marseille, France), ogen, Stem cell Technologies (U.S.A), Cellpro (USA) and Miltenyi Biotec GmbH (Germany). Alternatively, antibodies can be biotinylated or ated with digoxigenin and used in conjunction with avidin or anti—digoxigenin coated affinity columns.

According to an embodiment, different depletion/separation methods can be combined, for example, magnetic cell sorting can be combined with FACS, to increase the separation y or to allow sorting by multiple parameters.

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

T cell debulking may be effected using dies, including e.g. anti-CD8 antibodies, anti—CD4 antibodies, D3 antibodies, anti-CD2 dies, anti-TCRa/B antibodies and/or CRy/S antibodies.

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

B cell debulking may be effected using antibodies, including e.g. anti—CD19 or anti-CD20 antibodies. Alternatively, debulking in—vivo of B cells can be attained by on of anti-CD20 antibodies.

Alternatively, ve selection of CD34+ or CD131+ stem cells may be carried out using c. g. magnetic cell separation techniques (e. g. MACSTM), FACS sorter and/or capture ELISA labeling as bed in further detail above.

Following are a number of non-limiting es for depleting populations of cells of interest fe.g., T and/or B cells) from the immature hematopoietic cells according to the present invention prior to transplantation f: I. Separation of activated regulatory T helper cells following the teachings of PCT publication no. W0 2007/110249 and U.S. Patent no. 8,129,126 which are hereby incorporated by reference in their entirety: ing to one embodiment, there is provided a method of separating activated regulatory CD4+ CD25+ T helper (Th) cells from a cell preparation (e.g. immature hematopoietic cells) by use of the 4-1BB receptor.

The regulatory cells can be fied and/or separated through the expression of one and/or more markers. It is possible to employ for this purpose any marker known to the skilled worker for fication and/or separation. Exemplary markers are 4—1BB, CD25, CTLA~4 (cytotoxic T lymphocyte antigen~4), GITR (glucocorticoid—induced TNF receptor), FoxP3, IL—lO, CD69, CD4OL, ICOS, 0X40 and a, which are employed singly and/or in combination. It is possible for this purpose also to employ all markers known to the skilled worker for exclusion or depletion of non-regulatory cells in ation. However, in a particular embodiment, the 4—lBB receptor (CD137) is used as a marker of live, activated regulatory cells. 11. Separation of T regulatory cells or non-regulatory T cells ing the teachings of US. Patent Application No. 20110097313 which is hereby incorporated by reference in its entirety: According to one embodiment, there is provided a method of separating regulatory T cells (Tregs) or non—regulatory T-cells (conventional T—cells) from a cell preparation (e.g. immature hematopoietic cells) by the use of the CD154 molecule [CD40 ligand (CD4OL)].

As activated Treg cells D25+ Treg) do no express CD154, the present invention contemplates separation between activated (e. g. antigen activated) conventional T cells (CD154+ cells) and Treg (CD154- cells) using an agent capable of recognizing CD154 (e. g. anti—CD154 dy).

Thus, according to one embodiment, the present invention provides a method of separating activated Treg cells from a cell preparation (e. g. immature hematopoietic cells) using an agent capable of recognizing CD154. The present invention further contemplates the use of additional markers that are specific for regulatory T cells, such as for example, CD25, GITR, CTLA4, or markers which are specific for activated regulatory T cells, such as, for example, CD137, "latent ta (LAP)", GARP (LRRC32), CD121a/b for positive selection of Tregs.

According to another ment, there is provided a method of ing activated conventional T helper (Th) cells from a cell preparation (e.g. immature hematopoietic cells) using the CD154 marker. Specifically, using the CD154 marker, activated conventional Th cells (CD154 expressing cells) can be removed from a cell ation (comprising tory T cells), in ular, when additional selection methods using markers for activated/non—activated tory cells (CD137 and CD25, respectively) are used simultaneously or subsequently, which allows for use of the invention for fying isolating antigen—specific regulatory Th cells.

Thus, after obtaining a cell preparation (e. g. immature hematopoietic cells) from a donor and/or a subject, regulatory T—cells can be enriched using, for e, CD25.

These cells can be subsequently stimulated with a particular n. For this purpose, antibodies, peptides, proteins, chemicals substances (or mixtures thereof), or pathogens or cells may be used. After a particular activation time, the tory Th cells are identified and/or separated, for example, through the use of the CD137 marker. Thereby, antigen-specific regulatory Th cells are preferably identified and/or separated.

PCT/ILZOIZ/050541 III. Segaration at n-sgecific T cells following the teachings at US.

Patent 120. 084 which is hereby incorgorated by reference in its entirety: According to one embodiment, there is ed a use of CD154 [CD40 ligand (CD40L)] for the isolation of antigen—specific T cells wherein the cell preparation (e.g. immature hematopoietic cells) is ted with a CD40/CD154 system inhibitor.

According to an embodiment of the invention, the T cells are T helper (Th) CD4+ or CD8+ Th lymphocytes.

According to an embodiment of the invention, inflammatory, anti—inflammatory, regulatory and/or suppressive T cells are ed and/or ed.

The invention also relates to a method for the isolation of antigen-specific T cells in a cell preparation (eg. immature hematopoietic cells) following activation with an antigen, in which method the cell preparation (e.g. immature hematopoietic cells) is contacted with a CD40/CD154 system inhibitor, intra— and/or extracellular determination of CD154 is effected, and the cells having CD154 are isolated.

It will be appreciated that such ”contacting" with a CD40/CD154 system inhibitor of the cell preparation (e.g. immature hematopoietic cells) allows intra- and/or extracellular determination of CD154 and thus, isolation or separation of cells having CD154, the cells in particular representing the entire antigen-specific CD4+ Th lymphocytes.

Specifically, addition of a CD40/CD154 system inhibitor impairs or inhibits the interaction and signaling between CD40 and CD154. In the meaning of the invention, CD40/CD154 system inhibitors can be any of molecules or even physical exposures capable of ng or inhibiting the interaction n CD40 and CD154.

Accordingly, the inhibiting agent can be an antibody, e.g. one directed against CD40, or ed t CD154, a molecule, a cesium or lithium ion having an effect on the interaction between CD40 and CD154. The agent can also be a substance inhibiting the secretion or endocytosis in the cell, such as brefeldin A (Bref~A) and/or monsensin. Brefeldin A is a metabolite of the fungus Penicillium brefeldianum and, being a carboxylated ionophor, blocks the transport of newly synthesized ns from the endoplasmatic reticulum into the Golgi apparatus and impairs the exchange between endosomes and lysomes, while the circulation between cell membrane and mes advantageously remains undisturbed.

These substances ensure that CD40, CD154, the interaction between the two of them, or the CD40/CD154 system are modified in such a way that CD154 either is no longer downregulated and/or degraded on the cell surface, or, ed it is still within the cell, no longer transported therein. Such interruption of the transport within the cell prevents degradation of CD154. uently, CD154 is stabilized inside or outside the cell as an external receptor, thereby allowing detection and subsequent isolation using detection s well—known to those skilled in the art (explained in further detail herein).

IV. Separation of cells following the teachings of PCT publication no. WO 94/09117 and US. Patent no. 7,166,423 which are hereby incorporated by reference in their entirety: According to one embodiment, there is provided a method for tion of cells (6. g. B cells or T cells) from a cell preparation (e.g. immature hematopoietic cells) based on one or more products secreted from these cells (e.g. cytokines, including but not limited to, IFNy, 1L1, 1L2, 1L4, ILlO, IL12, TGF—beta, TNF, GM-CSF, and SCF, antibodies, hormones, enzymes and proteins). The method s products secreted by cells (e.g. B cells or T cells) to be captured at the surface of the cell (e.g. B cells or T cells). The captured product permits the cell (e.g. B cells or T cells) to be sorted according to the presence, absence, or amount of the product t. The means of capture comprise a capture moiety which has been anchored to the cell surface by a means le for the cell to be sorted.

The capture moiety may be coupled to the anchoring means (the ”anchor moiety") optionally h a linking moiety, and may also include a g moiety which multiplies the number of capture es available and thus the potential for capture of product, such as branched polymers, including, for e, modified dextran molecules, polyethylene , polypropylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone.

Suitable anchor moieties to the cell surface include lipophilic molecules such as fatty acids. Examples of suitable cell surface molecules include, but are not limited to, any molecule associated with the cell surface. Suitable les include, but are not limited to, cell surface markers such as CD45 (pan leukocyte), CD3 (T cells (activating)), CD4, CD8, CD19, CD20, CD14, CD16, CD15, class I MHC and Class II MHC molecules, CD34, CD38, CD33, CD56 T cell receptor, Fc receptor, betaZmicroglobulin or globulin, and other CD s or cell adhesion molecules. Alternatively, antibodies or other agents which specifically bind to cell surface les such as the MHC antigens or glycoproteins, could also be used.

Specific binding partners include capture moieties and label es. The capture moieties are those which attach both to the cell, either directly or indirectly, and the product. The label es are those which attach to the product and may be directly or ctly labeled. Specific binding partners include any moiety for which there is a relatively high affinity and specificity between product and its binding r, and in which the dissociation of the productzpaitner complex is relatively slow so that the productzpaitner complex is detected during the labeling or cell separation technique.

Specific label moieties may include, but are not limted to, fluorochromated anti- product antibodies, which may include, magnetic bead conjugated, colloidal bead conjugated, FITC, Phycoerythrin, PerCP, AMCA, fluorescent particle or liposome conjugated antibodies.

Specific binding partners may include, but are not limited to, substrates or substrate analogs to which a product will bind. These substrates include, but are not limited to, es, polysaccharides, steroids, biotin, digitoxin, nin, and other molecules able to bind the secreted product, and in a specific ment will include antibodies. When the capture moiety is an antibody it may be referred to as the re antibody" or "catch antibody.” As used herein, the term ”antibody" is intended to include polyclonal and monoclonal.

As used herein, the term ”antibody" is intended to include polyclonal and monoclonal antibodies, chimeric antibodies, haptens and antibody fragments, bispecific antibodies and molecules which are antibody equivalents in that they specifically bind to an epitope on the product antigen. Bispecific antibodies, also known as bifunctional antibodies, have at least one antigen recognition site for a first antigen and at least one n recognition site for a second antigen. Such antibodies can be produced by recombinant DNA s or chemically by methods known in the art. Chemically created bispecific antibodies include but are not limited to antibodies that have been WO 2013093919 reduced and reformed so as to retain their bivalent characteristics and antibodies that have been chemically d so that they have at least two n recognition sites for each antigen. Bispecific dies include all antibodies or conjugates of antibodies, or polymeric forms of antibodies which are capable of recognizing two different antigens.

Antibodies can be immobilized on a polymer or particle.

According to an embodiment, the capture moiety can be attached to a cell membrane by a variety of methods. Suitable s include, but are not limited to, direct chemical coupling to amino groups of the protein ents; coupling to thiols (formed after reduction of disulfide bridges) of the protein components; indirect coupling through antibodies (including pairs of antibodies); anchoring in the lipid bilayer by means of a hydrophobic anchor moiety; and g to the vely charged cell suxface by tions.

In other embodiments of the invention, the capture moiety is introduced using two or more steps, e.g., by labeling the cells with at least one anchor moiety which allows the coupling of the capture moiety to the anchor moiety either directly for ce by a biotin/avidin complex or indirectly through a suitable linking moiety or moieties.

Methods for direct al coupling of antibodies to the cell surface are known in the art, and include, for example, coupling using glutaraldehyde or maleimide activated antibodies. Methods for chemical coupling using multiple step procedures include, for e, biotinylation, coupling of TNP or digoxigenin using, for example, succinimide esters of these compounds. ylation may be accomplished by, for example, the use of D—biotinyl—N—hydroxysuccinimide. Succinimide groups react effectively with amino groups at pH values above 7, and entially between about pH 8.0 and about pH 8.5. Biotinylation may also be accomplished by, for example, treating the cells with dithiothreitol (DTT) followed by the addition of biotin maleimide.

Coupling to the cells may also be accomplished using antibodies against cell surface antigens ("markers"). Antibodies generally directed to sulface ns may be required in the range of about 0.1 to 1 ug of antibody per 107 cells, however, this requirement will vary widely in response to the affinity of the antibody to the product and will need to be determined empirically. Such a determination is well within the skill of one in the alt. Thus, the appropriate amount of antibody must be determined empirically and is within the skill of one in the art. This allows coupling to specific cells on cell type c marker expression. For instance, s of cells based such as T cells or subsets thereof can be specifically labeled. As a capture moiety, a bispecific antibody may be used which has an antigen ition site for the cell or an anchor moiety placed thereon, and the product.

A capture moiety, ularly capture antibodies should be selected based on the amount of secreted product. For example, for cells which secrete only a few les, a high affinity antibody should be chosen so as to catch most of the secreted molecules.

Alternatively, in the case where the cell secretes many molecules during the incubation time, a lower affinity antibody may be preferred to prevent too early saturation of the catching matrix. Determination of suitable affinities for the level of proteins secreted is determined cally and is within the skill of one in the art.

In some embodiments of the ion, the capture moiety is coupled to the cell by hydrophobic anchor moieties to the cell membrane. Suitable hydrophobic anchor moieties that will ct with the lipid bilayer of the membrane are known in the art, and include, but are not limited to, fatty acids and non-ionic detergents (including, e. g., Tween-80). A drawback to attachment of the capture moiety to the cell via the insertion of an anchor moiety is that the rate of integration of the anchor moiety into the cell is low. Thus, high concentrations of the anchor moiety often are required. This latter situation is often uneconomical when the capture moiety is a vely limited or ive substance, for example, an antibody. The low yield of hydrophobic anchor es that embed themselves in the membrane is relevant only when these les are available in relatively limited quantities. This problem may be overcome by using a bridging system that includes an anchor moiety and a capture moiety, wherein one of the moieties is of higher availability, and wherein the two parts of the bridging system have a high degree of specificity and affinity for each other. For example, in one embodiment, avidin or streptavidin is attached to the cell surface via a hydrophobic anchor , while the capture moiety is a biotinylated anti—product dy.

In another embodiment, the cell surface is labeled with digoxigenin followed by bispecific antibodies having specificity for both digoxigenin and the product. This approach can be used with other pairs of molecules able to form a link, including, for example, hapten with antihapten antibodies or NTA with polyhistidine residues. A specific embodiment is one which allows "amplification" of the system by increasing the number of capture moieties per anchor moiety.

In one illustrative embodiment, a ed n is bound to palmitic acid, thus providing a multiplicity of available binding sites. The dextran is in turn coupled to biotin and treated with avidin—conjugated antibody specific for the product. It is of course contemplated within the embodiments of the invention that linker moieties may be used between the anchor moiety and the capture moiety when the anchor moiety is coupled in any fashion to the cell surface. Thus, for example, an avidin (or streptavidin) biotin linker moiety may link an antibody anchor moiety with a capture moiety.

Bispecific antibody systems may also act as linker moieties.

In order to select cells that have the capability of secreting the product of interest, cells modified as above to contain the capture moiety are incubated under conditions that allow the production and secretion of the product in a sufficient amount to allow binding to and detection of the cells that n the captured product. These conditions are known to those of skill in the art and include, inter alia, appropriate temperature, pH, and concentrations of salts, growth factors and substrates in the incubation medium, as well as the appropriate concentrations of gas in the gaseous phase. When it is ble to distinguish between high and low producer cells, the time of incubation is such that product secretion by the cells is still in a linear phase. The appropriate conditions can be determined empin'cally and such a determination is within the skill of one in the alt.

Additionally, secretion by the cells can be d, that is upregulated, induced, or reduced using a biological modifier. Suitable ical modifiers include, but are not limited to, molecules and other cells. Suitable molecules include, but are not d to, drugs, cytokines, small molecules, hormones, combinations of eukins and other stimulating agents e.g. bispecific antibodies and other agents which modify cellular functions or protein expression. Other cells include, but are not limited to, direct cell to cell interactions such as between a tumor and T cell and indirect cell to cell interactions such as those induced by the ity of other cells which secrete a ical modifier. Suitable cells include, but are not limited to, blood cells, peripheral bone marrow cells (PBMC) and various cell lines. The ical rs can be added at any time but are preferably added to the incubation medium.

PCT/11.2012/050541 Alternatively, the cells can be pretreated with these agents or cells prior to the incubation step. The incubation ions are also such that product secreted by a producer cells is essentially not captured by another cell, so guishing non- producing cells from product producing cells, or high producers from low producers is possible. lly the incubation time is between 5 minutes and ten hours, and more usually is between 1 and 5 hours. The incubation medium may optionally include a substance which slows diffusion of the ed product from the producer cell.

Substances which inhibit product diffusion in liquid media and that are non—toxic to cells are known in the art, and include, for example, a y of substances that partially or completely gel, including, for example, alginate, low melting agarose and gelatin.

By varying the viscosity or permeability of the medium, the local capture by a ing cell of certain sizes of secreted products can be modulated. The molecular weight size exclusion of the medium can be adjusted to optimize the reaction. The optimal composition of the medium can be empirically determined and is influenced by the cell concentration, the level of secretion and lar weight of the product and the affinity of the capture antibodies for the product. Such a determination is within the skill of one in the art.

Preferably, the gels are solubilized after the incubation to allow for the isolation of the cells or groups of cells from the media by cell sorting techniques. Thus, for example, the gels may be linked by de bonds that can be iated by dryl reducing agents such as /3—mercaptoethanol or DTT or the gels may contain ionic cross— linkings, including for example, calcium ions, that are solubilized by the addition of a chelating agent such as EDTA.

In a ic ment, during the secretion phase, the cells are incubated in a gelatinous medium, and preferentially the size limitation of penetration into the gel prevents the product from substantially entering the gel.

An alternative or addition to using a viscous or gelatinous medium to prevent unspecific cell contamination is to provide a capture system for capturing products not captured by the cell surface capture system on the secreting cell. For example, this que can be used in the case where many cell types produce a product or if no sufficient diffusion barrier can be created between the cells. This can be accomplished by adding to the medium surrounding the cells beads (e.g. latex beads) conjugated to an antibody product from the supernatant. Alternatively, gels with immobilized antibodies or other moieties being able to remove d product from the medium might be employed. These trapping moieties are capable of retaining these unwanted products or preventing them from binding to the non—secreting cells by binding to the non-retained products. This "junk capture system" might consist of immobilized into the gel matrix or it may be ed to magnetic or other types of particles. The location and catching characteristics of the junk capture system should be ed so that sufficient product les are specifically bound to the secretng cells thus minimizing background on non—producing cells.

At the end of the secretion phase the gel matrix (if any) is solubilized. The cells containing the captured product are then d with a label moiety. Labeling may be accomplished by any method known to those of skill in the art. For example, anti— product antibodies may be used to directly or indirectly label the cells containing the product. The labels used are those which are suitable for use in systems in which cells are to be ed or sorted based upon the attachment of the label moiety to the In other embodiments, capture moieties that do not contain captured product may be detected. This allows, for example, the isolation of cells that secrete high amounts of product by employing a negative tion method, i.e., detection of cells not highly saturated with product. The cells can be d with other substances recognizing, including, but not limited to, cell e markers, cell type, cellular parameters such as DNA content, cell status, or number of capture moieties.

The enumeration of actual capture moieties can be important to compensate for varying amounts of these molecules due to, for example, different conjugation potentials of the cells. It may be especially important for the isolation of rare cells to exclude cells with decreased or increased capability for binding the product capture system, ing the anchor and capture moieties.

Analysis of the cell population and cell sorting based upon the presence of the label may be lished by a number of techniques known in the art and are described in r detail herein.

W0 2013I093919 V. Separation of aiztigen-sgecific T cells following the teachings 0t US.

Patent no. 6,576,428 which is hereby incorporated by reference in its entirety: According to one embodiment, there is provided a method for cell separation of antigen-specific T cells from a cell preparation (e.g. immature hematopoietic cells) based on one or more products secreted by these T cells (e.g. cytokines or growth factors, including but not limited to, IL-3, GM—CSF, IL—2, IFN—gamma, TNF—alpha, IL— 4, IL—5, IL—10 and/or IL—13) in response to n stimulation. The T cells are provided with a capture moiety (e. g. antibody) for the product, which can then be used directly as a label in some instances, or the bound product can be further labeled via label moieties (i.e. detectable ) that bind specifically to the product and that are labeled with traditional labeling materials such as fluorophores, radioactive isotopes, chromophores or magnetic particles. The labeled cells are then separated using standard cell sorting techniques based on these labels. Such techniques include flow cytometry, magnetic gradient separation, fugation, and the like (as described in further detail ).

According to one embodiment, antigen ation is achieved by exposing the cell preparation (e. g. immature hematopoietic cells) to at least one antigen under conditions effective to elicit antigen—specific stimulation of at least one T cell. ng with the product is achieved by ing the surface of the cells to contain at least one capture moiety, culturing the cells under conditions in which the product is secreted, released and cally bound ("captui‘ed" or "entrapped") to the capture moiety; and labeling the ed product with a label moiety, where the d cells are not lysed as part of the labeling ure or as part of the separation procedure.

According to another embodiment, cell preparation (e. g. immature hematopoietic cells) can be further subjected to one or more separation protocols based on the expression of cell surface markers. For example, the cells can be subjected to positive selection on the basis of expression of one or more cell e polypeptides, including, but not limited to, "cluster of entiation (CD)" cell surface markers such as CD2, CD3, CD4, CD8, TCR, CD45, CD45RO, , CDllb, CD26, CD27, CD28, CD29, CD30, CD31, CD4OL; other markers associated with lymphocyte tion, such as the lymphocyte activation gene 3 product (LAG3), signaling lymphocyte activation molecule (SLAM), Tl/ST2; chemokine receptors such as CCR3, CCR4, CXCR3, W0 20131093919 2012/050541 CCRS', homing receptors such as CD62L, CD44, CLA, CD146, alpha.4.beta.7, alpha.E.beta.7; activation s such as CD25, CD69 and 0X40; and lipoglycans presented by CD1. Alternatively, the cell preparation (e.g. immature hematopoietic cells) can be further subjected to ve selection for depletion of non-T cells and/or particular T cell subsets. Negative selection can be performed on the basis of cell surface expression of a y of molecules, including, but not limited to, B cell markers such as CD19, and CD20; monocyte marker CD14; the NK cell marker CD56.

As mentioned, T cell or B cell debulking may be ed in-vitro or in-vivo (e.g. in a donor piior to ing immature hematopoietic cells therefrom).

Following are a number of non-limiting examgles [or g 178 cell debulking according to the gresent invention. 1. MACSTM following the teachings of PCT ation no. W0 2009/066180 which is hereby incorgorated by nce in its entirety.

According to one embodiment, debulking in vitro of T or B cells (TCD and/or BCD) is effected by magnetic—activated cell sorting (MACSTM). Thus, MACSTM provides means for the separation of a particular living cell from a population of living cells.

The cell separation is typically performed by adding an antibody to the 2O population of cells that specifically binds to an extracellular epitope of a surface marker (in this case T/B cell marker e.g. r of differentiation, i.e. CD, polypeptide). It is advantageous that the antibody is labeled with a detectable agent suitable for cell sorting (cell separation), such as a fluorescent dye (e.g. FITC) or a magnetically responsive agent (e.g. bead).

Thus, the antibody may be coupled to a magnetically responsive agent, whereupon the antibody can be used to separate the cell bound to it under conditions sufficient to specifically bind the antibodies to the epitope (antigen).

According to an embodiment, and as taught in WO/2009/O66180, the method r comprises the following steps: immobilizing the cell expressing the surface marker that is specifically bound to the antibody labeled with a magnetically responsive agent in a ferromagnetic matrix (described in further detail hereinbelow) through a magnetic field; washing the matrix to remove unbound cells; and removing the magnetic W0 2013/‘093919 field to elute the cell from the matrix. Thereby, a cell sample enriched in or consisting of cells expressing the e marker is provided.

The elution of the ferromagnetic matrix can be performed using gravity flow, centrifugation, vacuum filtration or by positive pressure, e.g. using a plunger.

The term "magnetic cell sorting" as used herein refers to procedures for cell separation (cell g) including, but not limited to, magnetic separation using antibodies linked to colloidal magnetic particles, affinity chromatography and cytotoxic agents joined to a monoclonal antibody or used in conjunction with any antibody— dependent separation technique known in the art.

In an exemplary ment, monoclonal antibodies are used in conjunction with dal supeiparamagnetic microparticles having an organic coating by e.g. polysaccharides (Miltenyi et al. (1990) Cytometry 11:231—238). These particles can be used having a size of 10 to 200 nm, ably between 40 and 100 nm, and can be either directly conjugated to antibodies or used in combination with anti-immunoglobulin, avidin or anti-hapten—specific microbeads. Polysaccharide—coated superparamagnetic particles are commercially available from Miltenyi Biotec GmbH, Germany.

Methods to prepare superparamagnetic particles as described in US. Pat. No. 4,770,183 can be combined with the t teachings.

With respect to terminology, as is the general usage in the art: As used herein, the term "ferromagnetic" relates to als which are strongly susceptible to magnetic fields and are capable of retaining magnetic properties when the field is d. Ferromagnetism occurs only when ed electrons in the mateiial are contained in a crystalline lattice thus permitting coupling of the unpaired electrons.

As used herein, the term "superparamagnetic" relates to materials which are highly magnetically susceptible, i.e., they become strongly magnetic when placed in a magnetic field, but, rapidly lose their ism. Superparamagnetism occurs in ferromagnetic materials when the crystal diameter is decreased to less than a critical value.

It will be appreciated that the extent of magnetization which is acquired by a particle is a function of its ic susceptibility and the applied magnetic field. The magnetization is a function of the resulting magnetic moment and of the volume of the particle. The higher the magnetic moment and the smaller the , the higher the magnetization.

II. MACSTM following the teachings of U.S. Patent No. 5,411,863 which is hereby incorgorated by nce in its entirefl.

According to one embodiment, and as taught in U.S. Patent No. 5,411,863, the cell separation [debulking in vitro of T or B cells (TCD and/or BCD)] may be carried out in a high gradient magnetic separation (HGMS), namely by a procedure for selectively retaining magnetic materials or gnetic targets labeled with magnetic particles in a chamber or column disposed in a magnetic field. Thus, for example, the fluid containing the magnetic particles is passed through a vessel or column which is ed in a magnetic gradient. In desirable ways to conduct cell separations, the vessel is filled with a matrix which is capable of creating high magnetic gradients in the neighborhood of its surface. While the strength of the magnetic field imposed on the particles determines their ization, their retention is a function of the strength of the magnetic gradient.

Magnetized particles are retained by high magnetic gradients. Typical matrices are filamentous or particulate metals such as steel wool, wires or filaments or particulates or grids.

The present invention r provides a method of coating such matrices which both ntly and effectively ts biological materials subjected to passage through the matrix from damage which would be caused by exposure of these materials to the ic surface. The coating on the matrix effectively prevents the corrosion of the metallic surfaces and prevents the passage of any ions which might form at the surface into the surrounding fluid. Furthermore, the impermeable coating provided by the invention adds physical stability to the matrix. onal publications describing a variety of HGMS systems are known in the art, and include, for example, U.S. Patent No. 4,452,773, U.S. Patent No. 4,230,685, PCT application WO 85/04330, U.S. Patent No. 4,770,183, and PCT/EP89/01602; systems are also described in U.S. Serial No. 07/291,177 and in U.S. Serial No. ,176, all of which are incorporated herein by reference.

III. Magnetic segaration (MACSTMQ aggamtus ing the teachings of US. Patent Nos. 5,786,161 and 5,711,871 which are hereby incorgorated by reference in their ty.

According to one embodiment, a magnetic separation device may used in line with the present ion.

According to one embodiment, a magnetic separation device for magnetic separation procedures contains matrices which provide uniform pores or channels that reduce the entrapment of air or non-target substances, and decrease the loss of target substances due to mechanical disruption. Target cells (e.g. B or T cells), from various biological samples are labeled in ction with a suitable specific binding member (e.g. T/B cell specific dy, as described above), and isolated using the devices and methods of the present invention.

The separation system is able to cally select and separate a defined population of cells (target cells) from a mixed cell population, such as peripheral blood, bone marrow, blood from the umbilical cord or placenta, fetal blood or a leukapheresis According to one embodiment, a high gradient magnetic separation (HGMS) device is used in line with the present ion.

Conventional high gradient magnetic separation matrices are lly prepared from materials such as wires, metal—coated fiber or steel wool. In the improved magnetic separation device of the present invention, the gradient—intensifying matrix of the high gradient magnetic separator is formed from small spheres of magnetically tible or ferromagnetic material. Such materials include, but are not limited to iron, steel, cobalt nickel, and other ferromagnetic rare earth metals or alloys thereof. For e, the matrix material may e ferromagnetic metal spheres such as iron spheres (e.g.

MARABU Balls, Kugelfabrik Schulte & Co., Wermelskirchen, Germany).

Many different methods of manufacturing spheres are known. Usually the spheres have an average diameter ranging from about 0.2 to 1.5 mm for the separation of large cells or cell complexes, and about 0.05 to 0.2 mm diameter for subcellular material. Specifically, the spheres have an average diameter g from about 0.2 to 0.5 mm, and most ically, the spheres are selected to have an average diameter ranging from about 0.2 to 0.3 mm. It is desirable that the size of spheres be relatively neous, usually varying not more than about 15% from the average size, more usually by not more than about 10%, and preferably by not more than about 5%.

The spheres are composed of a ferromagnetic al (e.g. iron, steel, etc.), which may be coated with an impermeable coating to prevent the t of cells with metal. By eable coating it is meant a polymeric coating which contains substantially less than 30% water by weight, which does not permit the passage of ions, and which is feimed on the sphere as a result of passive application, cross—linking or polymerization of a relatively hydrophobic polymer or co-polymer. Suitable polymers include polystyrenes, polyacrylamides, polyetherurethanes, polysulfones, nated or chlorinated polymers such as polyvinyl chloride, polyethylenes and polypropylenes, polycarbonates and ters, etc.

The matrix of spheres should have adequate surface area to create sufficient magnetic field gradients in the separation device to permit efficient retention of magnetically labeled cells. The volume necessary for a given separation may be empirically determined, and will vary with the cell size, antigen density on the cell surface, antibody affinity, etc. The flow rate will be determined by the size of the , but will lly not require a cannula or valve to regulate the flow.

The labeled cells are retained in the magnetic tion device in the presence of a magnetic field, usually at least about 100 mT, more usually at least about 500 mT, usually not more than about 2 T, more usually not more than about 1 T. The source of the magnetic field may be a permanent or electromagnet. After the initial binding, the device may be washed with any suitable physiological buffer to remove unbound cells.

The bound cells are released from the ic separation means by removing the magnetic field, and eluting in a suitable buffer. The cells may be collected in any appropriate , preferably one that maintains the viability of the cells. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, PBS—EDTA, PBS, Iscove's medium, etc., which may be supplemented with fetal calf serum, BSA, HSA, etc. The cells may then be used as appropriate and as taught herein.

In its simplest f01m, the cell tion system of the present invention has two main components: a magnetic separator and a cell separation reagent. A more complex separation device includes fluid passages, collection and storage containers and the separation column. The fluid circuitry can be constructed with ated valves, or the valves may be applied externally to the fluid pathways Additional magnetic separation devices which may used in line with the present invention are described in WO/90/O7380, PCT/US96/00953 and EP 438,520, US ,779,892, US 6,417,011 all of which are incorporated herein by nce.

IV. MACSTM following the teachings at US. Patent No. 6,900,029 which is hereby incorgorated by reference in its entirefl. ing to another embodiment of the present invention, a population of cells may be selected by utilizing les and gravity sedimentation as taught in US. Patent No. 6,900,029. Thus, T/B cell ing may be carried out by removal of an undesired population or subpopulation of cells (e.g. cells expressing CD20, CD19, CD2, CD4, CD8) by separating f from a biological sample (e. g. fluid sample), such as whole blood or bone marrow, y and with a high yield using a plurality of dense, relatively heavy particles having one or more reactants, such as monoclonal or polyclonal dies, bound o mixed with the biological sample (e. g. fluid sample). The antibodies bound to the particles can be directed at the T or B cells which are to be removed from the biological sample. The particles with the cells bound thereto are allowed to differentially settle by gravity and then the remaining sample ising the T/B cell depleted sample e.g. immature hematopoietic cells) is removed for further use. This enhances the number of remaining cells of interest (e. g. immature hematopoietic cells) in the sample which were not targeted by the particles. Accordingly there is provided a high yield of the cells of interest even after multiple removal steps.

The antibodies bound to the particles also can be directed at the cells of interest (e.g. immature hematopoietic cells expressing, for example, CD34). The targeted cells (e.g.

CD34+ immature poietic cells) can then be removed from the particles for further use.

According to one embodiment, the particle material can be nickel. The nickel le can be heated to sterilize the particle where desired. If the sample has been purged and is to be transplanted into a human, a magnetic field and washing procedure can be utilized (as described in detail above) to remove r undesired cells (e. g. red blood cells) and to further ensure that all the dense particles have been removed from the biological sample.

According to one embodiment, the T cell depleted immature hematopoietic cells (e. g. comprising CD34+ cells) comprise T cell depleted bone marrow cells, T cell ed mobilized peripheral blood progenitor cells (e. g. mobilized by G—CSF), T cell depleted cord blood/fetal liver/yolk sac and/or, purified CD34+ cells (harvested from all the sources ned above e.g. from bone marrow and/or from G—CSF mobilized peripheral blood progenitor cells) and selected by positive selection (e.g. with magnetic beads using an D34 antibody, e.g. as bed above using MACSTM). 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 t 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, 8 x 106, 8.5 x 106, 9 x 106, 9.5 x106, 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 kilogram body weight.

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

According to a specific embodiment, the subject is administered a dose of T cell ed immature poietic 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 hematopoietic cells comprising a range of about 4—30 x 106, 4—40 X 106, 4-50 x 106, 4—60 x 106, 4-70 x 106, 4-80 x 106, 4-90 x 106, 4100 x 106, 5-10 x 106, 5- 20 x 106, 5-30 x 10‘15-40 x 106, 5-50 x 106, 5-60 x 106, 5—70 x 106, 5—80 x 106, 5-90 x 106, 5100 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 106, 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, 40-100 x 106, 50-60 x 106, 50-70 x 106, 50-80 x 106, 50-90 x 106, 50-100 x 106, 60-70 x 106, 60—80 x 106, 60-90 x 106, 60-100 x 106, W0 93919 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 stered a dose of T cell depleted immature hematopoietic cells comprising 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 may 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 ned, the subject of the instant invention may further be transplanted with a cell or tissue graft (e. g. liver, as, 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 recipient's disease (e.g. organ failure); the physical or physiological ters c to the t; 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 application. The cell or tissue graft may be transplanted into a homotopic anatomical location (a normal anatomical location for the transplant), or into an ectopic anatomical location (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 testicular fat, the sub cutis, the m, 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 present teachings may be transplanted into the liver, the portal vein, the renal capsule, the sub-cutis, the omentum, the spleen, and the intra-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 hepatic transplantation (cg. hepatic failure). rly, transplanting a pancreatic tissue according to the present ion may be advantageously effected by transplanting the tissue into the portal vein, the liver, the pancreas, the ular 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 (e.g. diabetes). Likewise, transplantation of tissues such as a kidney, a heart, a lung or skin tissue may be carried out into any anatomical location described above for the purpose of treating recipients suffering from, for example, renal failure, heart failure, lung failure or skin damage (e.g., burns).

Optionally, when transplanting a cell or tissue graft of the present ion into a t 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 structural/functional integration thereof with the anatomy/physiology of the t.

The method of the present invention also ons nsplantation of l organs (e.g. heart and lung, liver and spleen, pancreas and bone marrow e.g. hematopoietic stem cells, kidney and bone marrow e. g. hematopoietic stem cells, etc.) in case the subject may be beneficially effected by such a procedure.

According to one embodiment, the co—transplantation ses lantation of immature hematopoietic cells and a solid tissue/organ or a number of solid organs/tissues.

According to one embodiment, the immature hematopoietic cells and the solid organ are obtained from the same donor. ing 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. comprising 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 rd art techniques. For example, the functionality of a pancreatic tissue transplant may be red following transplantation by standard pancreas function tests (e.g. analysis of serum levels of insulin). Likewise, a liver tissue transplant may be monitored following transplantation by standard liver function tests (e. g. analysis of serum levels of albumin, 2012/050541 total n, ALT, AST, and bilirubin, and analysis of blood-clotting time). Stiuctural 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 %, 60 %, 70 %, 80 %, 90 % or 95 %, or ably avoid graft rejection and/or graft versus host disease (GVHD), the present invention contemplates post transplant stration of cyclophosphamide.

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

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

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 ic ment, the cyclophosphamide refers to the molecular formula C7H15C12N202P°H20 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 (Geiman Remedies), Roxane Laboratories Inc-Boehn'nger eim, Bristol—Myers Squibb Co ~ Mead Johnson and Co, and Pfizer — Pharmacia & Upjohn, under the brand names of Endoxan, Cytoxan, Neosar, Procytox and Revimmune.

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

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

For example, in case of cell or tissue graft, the eutic effective amount of cyclophosphamide comprises about 1-25 mg, l-50 mg, 1—75 mg, 1—100 mg, 1—250 mg, 1-500 mg, 1-750 mg, 1-1000 mg, 5-50 mg, 5-75 mg, 5-100 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, lO—lOOO mg, 25—50 mg, 25—75 mg, 25-100 mg, 25~125 mg, 25—200 mg, 25—300 mg, W0 20131093919 PCT/11,2012/050541 —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, 5 mg, 100-150 mg, 100-200 mg, 100-300 mg, 0 mg, 100-500 mg, 00 mg, 125-150 mg, 125- 250 mg, 125—500 mg, 125-1000 mg, 150—200 mg, 150—300 mg, 150—500 mg, 00 mg, 200—300 mg, 200—400 mg, 200—500 mg, 200~750 mg, 200—1000 mg, 250—500 mg, 250—750 mg, 250—1000 mg per am body weight of the subject.

According to a ic embodiment, the therapeutic effective amount of cyclophosphamide is about 25-200 mg per kilogram body weight of the subject.

As illustrated in the Examples section which follows, the present inventors have shown that stration of two doses of cyclophosphamide post transplant (on days 3 and 4 post transplant) allows for a durable 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, cyclophosphamide 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 stered daily such as once a day or twice a day.

The dose of each cyclophosphamide administration may se 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 of cyclophosphamide 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 subject 1, 2, 3, 4, 5, 6, 7, 8, 9, days or more post transplant (i.e., T+l, +2, +3, +4, +5, +6, +7, +8, +9, +10).

According to a specific embodiment, cyclophosphamide is stered to the subject in two doses 3 and 4 days post lant.

According to an embodiment, hosphamide is stered prior to transplantation and post transplantation. Thus, for e, cyclophosphamide may be administered to the subject 3 days prior to lantation (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 transplantation and the subject's response to the regimen. ination 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 conditioning the t 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 (e.g. cyclosporine A, as bed in further detail below) in order to avoid organ rejection. Once the T cell depleted immature hematopoietic cells are transplanted and chimeiism is achieved, the general immune suppression agents may be tapered down and subsequently stopped. In st in situations in which the cell or tissue graft (e.g. solid organ) is transplanted subsequent 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 regimens for transplantation is provided in the literature of the art (for e, 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 al., 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 ioned under reduced intensity conditioning prior to transplantation of a cell or tissue graft.

According to an ment, the reduced intensity conditioning is effected for up to 2 weeks (e.g. 1-10 or 1—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 descn'bed in detail in the Examples section which follows, in-vivo T cell debulking e. g. by anti—CD4 antibody, 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 eutic ive dose of about 300 pg 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.

Thus, according to one embodiment, the TBI comprises a single or fractionated 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, 1—7.5 Gy, 1'10 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—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 ic embodiment, the TBI compiises a single or fractionated in‘adiation dose within the range of 1-3.5 Gy.

According to an embodiment, TBI treatment is stered to the subject 1—10 days (6. g. 1—3 days) prior to transplantation. According to one embodiment, the subject is conditioned once with TBI 1 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, 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, 1-10 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 Cry, 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— Gy, 10—40 Gy, 10—50 Gy, 0.5-20 Gy, 0.5—30 Gy, 0.5—40 Gy or 05-50 Gy.

According to a specific embodiment, the TLI comprises a single or fractionated irradiation dose within the range of 1-3.5 Gy.

According to an ment, 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 ioned 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 s may be administered to the subject in a single dose or in several doses e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses (e.g. daily doses) prior to transplantation. According to one embodiment, the subject is administered a chemotherapeutic agent (e.g. Fludarabine e.g. at a dose of about 30 mg/mZ/day) for 5 consecutive days prior to lantation (6. g. on days —7 to —3).

According to one embodiment, the conditioning 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. Campath, MabCampath, Campath—lH and da) and an anti-thymocyte globulin (ATG) agent [e. g.

Thymoglobulin (rabbit ATG, rATG, ble from Genzyme) and Atgam (equine ATG, eATG, available from Pfizer)]. Additional antibody immunotherapy may comprise anti— CD3 (OKT3), anti—CD4 or anti—CD8 agents. 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 (6. g. daily doses) prior to transplantation (6. g. 6 days prior to transplantation).

According to one ment, the subject is not treated chronically (e.g. for a ged 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 . For example, the subject may be administered with graded doses of T-cells as WO 2013093919 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 t may be treated by infusion of about 0.5 — 5 x 104 CD3+ cytes per kg recipient body weight (e.g. l 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 logical relapse will further be treated with a first dose of about 1 x 104 CD3+ cells per Kg recipient body weight. In the absence of GVHD, the second infusion of about 1 x 105 CD3+ cells per kg recipient body weight will typically be given about 45 days later followed 2 months later by a third dose of about 1 x 106 CD3+ cells per kg ent body weight. It will be iated that donors typically undergo a leukoapheresis to collect lymphocytes prior to mobilization of hematopoietic cells (e. g. for transplantation). The frozen products are thawed as needed and infused quickly over a period of 5—10 s. ts exhibiting acute GVHD or who fail to trate hematological engraftment typically will not receive any DLI.

According to one embodiment, a patient with relapsing B cell non-Hodgkin lymphoma will typically be further treated with rituximab (cg. 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 11) before starting DLI.

According to one embodiment, no post—DLI immunosuppressive agents will be used along with the present methods.

According to an aspect of the t ion, there is provided a method of treating a subject in need of a T cell depleted re hematopoietic cell transplantation, the method comprising: (a) transplanting into a conditioned subject a dose of T cell depleted immature hematopoietic cells, wherein 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 eutically effective amount of cyclophosphamide, wherein the eutically effective amount comprises 25—200 mg per kilogram body weight, thereby treating the subject.

According to an aspect of the t invention, there is provided a method of treating a t in need of an immature hematopoietic cell transplantation, the method comprising: (a) conditioning a subject under a reduced intensity conditioning protocol, wherein the reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent; (b) lanting into the subject a dose of T cell depleted re hematopoietic cells, wherein the T cell depleted immature hematopoietic cells se 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 uently (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.

According to an aspect of the present invention, there is provided a method of inducing donor specific tolerance in 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 depleted immature hematopoietic cells obtained from a non-syngeneic donor, wherein the T cell depleted re 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) administeiing 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.

As used herein, the term “donor specific tolerance” as used herein refers to a ion 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 donor’s cells (e.g. donor hematopoietic cells) as compared to the responsiveness of the recipient's cells in the absence of such a treatment method. nce induction enables transplantation of a cell or tissue graft (as described in further detail hereinabove) with reduced risk of graft rejection or GVHD.

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

WO 2013093919 According to one embodiment of the present invention, DLI may comprise l X 103 - l 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 e, 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 hematological e may e total body irradiation (TBI), total lymphoid 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 itant with the second rituximab dose.

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

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

The terms "comprises", "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 ition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the d composition, method or ure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound” may include a plurality of compounds, including mixtures thereof.

WO 93919 Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and y and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, ption 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 s 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 or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first te number “to77 a second indicate number are used herein interchangeably and are meant to include the first and second ted numbers and all the fractional and integral numerals therebetween.

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, biological, biochemical and l 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 invention, which are, for brevity, described in the context of a single embodiment, may also be provided tely or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be ered essential features of those ments, unless the embodiment is inoperative without those elements. s embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

WO 93919 PCT/ILZOIZ/050541 EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and inant DNA techniques. Such techniques are thoroughly explained in the ture. See, for example, I'Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-lH Ausubel, R. M., ed. (1994); 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 is: A Laboratory Manual Series", Vols. 14, 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 Biology: A Laboratory Handbook", s I—III Cellis, J. 13., ed. (1994); "Current ols in Immunology” Volumes I-lII Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and al Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), ted Methods in Cellular Immunology", W. H. n and C0,, New York (1980); available immunoassays are extensively described in the patent and scientific literature, 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; 4,879,219; ,011,771 and 5,281,521; "Oligonucleotide Synthesis” Gait, M. J ., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J ., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. ; "Animal Cell Culture" Freshney, R. L, ed. (1986); ilized Cells and Enzymes" IRL Press, ; "A Practical Guide to lar Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1—317, Academic Press; "PCR ols: A Guide To s And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization — A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are ed for the convenience of the reader.

All the information contained therein is incorporated herein by reference.

EXAMPLE 1 Stable engrafiment 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. Balb/c—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 ed 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 source of BM depleted of T cells) were transplanted into allogeneic recipients (C3H/Hen) on day 0 following in—vivo T cell debulking (TCD) with anti—CD4 (clone GKl.5) and anti—CD8 (clone 53.6.72) antibodies (300 ug each; Bio X Cell, NH, USA) red on day —6, and exposure to 2.0 Gy total body irradiation (TBI) on day —l.

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

Skin rotocol Donor (Balb/c) and 3rd party (C57BL/6) skin grafts were transplanted to the mixed chimeric ents as described above {i.e. to those mice which were previously transplanted with mega dose (25 x 106) T cell depleted BM 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.

PCT/ILZOIZ/050541 RESULTS To test the potential y 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. ent 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 d 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, 100 mg/kg) was stered on days +3 and +4 post lant. 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 revealed that none of the l mice (conditioned with TCD, 2 Gy TBI and optionally CY, but did not receive BM) sed donor type chimerism. Similarly, none of the BM mice recipients that were transplanted with a regular dose of 5 x 106 T 2O cell depleted BM, in the presence or absence of cyclophosphamide treatment, expressed donor type chimerism. However when the dose of T cell ed BM was increased to x 106 cells, durable mix chimerism was achieved in 4 out of 7 mice that were also treated with cyclophosphamide on days +3 and +4 post transplant (see Figures 1A—B and Figure 2C).

Further -up of these recipient mice at 180 and 225 days post transplant revealed that the chimeiism d was stable and durable (Figure 3). As illustrated in Figure 3, the number of donor type chimeric ents and the level of donor chimerism remained unchanged 225 days post transplant, suggesting that tolerance has been achieved.

Tolerance 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 WO 2013093919 described above), in comparison to the recipients 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 (Figure 4A).

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

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

EXAMPLE 2 The effect of different doses of total body irradiation (TBI) on chimerism MATERIALS AND EXPERIMENTAL PROCEDURES Animals As described in Example 1, hereinabove. lantation protocol High dose (25 x 106) Balb/c—Nude BM cells (providing a source of BM depleted of T cells) were transplanted into 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 ent doses of irradiation g 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 (e.g. FlTC labeled anti-H—2Dd dy specific for donor type cells and PE labeled anti-H-ZKk antibody specific for host type cells). 2012/050541 RESULTS In this experiment, the minimal irradiation dose was defined. ‘Mega dose’ (25 x 106) Balb/c—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 ng from 1 to 3.5 Gy TBI) on day —1. High dose Cyclophosphamide (CY) was administered on days +3 and +4 post lant and donor type chimeiism was evaluated at 30 days post transplant.

As can be seen in Figure 5, all the recipient mice that were irradiated with 2.5, 3 or 3.5 Gy TBI (6/6) were chimeric, exhibiting donor type chimeiism ranging between 58 — 83 %. Similarly, 87 % (13/15) 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), r the donor type chimerism range was significantly reduced to 14.5 — 58 %.

EXAMPLE 3 The efiect ofdifferent Cyclophosphamide (CY) doses 0n chimerism MATERIALS AND EXPERIMENTAL PROCEDURES 2O Animals As described in Example 1, hereinabove.

Transplantation protocol High dose (25 x 106) Balb/c~Nude BM cells (providing a source of BM depleted of T cells) were transplanted into allogeneic recipients (C3H/Hen) on day 0 following in—vivo T cell debulking (TCD) with D4 (clone GK1.5) and anti—CD8 (clone 53.6.72) antibodies (300 ng each; Bio X Cell, NH, USA) delivered on day —6, and exposure to 2.0 Gy total body ation (TBI) on day —1. Different doses of Cyclophosphamide (CY, 100 mg/kg, 125 mg/kg or 150 mg/kg, Baxter Oncology, Germany) were administered on days +3 and +4 post transplant and donor type chimerism was evaluated 30 days post transplant using cein anti-host and donor H-2 dies (e. g. FlTC labeled anti-H-2Dd antibody specific for donor type cells and PE labeled -2Kk antibody specific for host type cells).

RESULTS In this experiment, the l dose of CY post lant was defined. ‘Mega dose’ (25 x 106) Balb/c-Nude BM cells were transplanted into 3 groups of allogeneic recipients (C3H/Hen) on day 0 following T cell debulking (TCD) with anti—CD4 and anti—CD8 antibodies on day —6, and 2 Gy TBI on day -1. Different doses of Cyclophosphamide (CY), 100 mg/kg, 125 mg/kg or 150 mg/kg, were administered on days +3 and +4 post lant 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 chimerism. Thus, the recipient mice that were treated with 100 mg/kg, 125 mg/kg or 150 mg/kg CY exhibited an average of 57.5 i .8, 66.5 i 20.6 or 67.4 :t 27.4 donor type chimerism, respectively. No statistical significance was found when the recipients treated with 100 mg/kg were compared to those treated with 125 mg/kg or 150 mg/kg (P205 and p=0.469 tively).

EXAMPLE 4 CD8+ Non-T cells are not importantfor attaining chimerism by combining ‘mega dose’ T cell ed BM with CYpost transplant MATERIALS AND EXPERIMENTAL PROCEDURES Animals As described 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 GKl.5) and D8 (clone 53.6.72) antibodies (300 pg each; Bio X Cell, NH, USA) delivered on day -6, and exposure to 2.0 Gy total body irradiation (TBI) on day ~l. High dose hosphamide (CY, 100 mg/kg, Baxter Oncology, Germany) was administered on days +3 and +4 post transplant and donor type chimerism was ted 30 days post lant using fluorescein anti-host and donor H-2 antibodies (6. g. FlTC labeled anti- H—2DCl 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 Balb/c—Nude mice. Moreover, the transplanted mice in these experiments were athymic 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 preparation from Balb/c-Nude mice was negatively sorted for CD8 cells using a cell sorting system (e.g. anti—CD8 magnetic beads or FACS sorter).

RESULTS As Hdstad et al. previously taught that a subset of CD8+ 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) —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 Transl Med. (2012) 4:1241‘al28], the present inventors depleted residual CD8+ cells from the Balb/c—Nude ‘mega dose’ BM ation, and measured Chimerism induction compared to control non—CD8+ depleted Nude BM cells.

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

EXAMPLE 5 alprotocol STUDY DESIGN This is a prospective, observational, phase I/II multicenter study. Ten patients with hematological disorders will be enrolled over a one year period.

The primary endpoint of the study is tment and 10 evaluable patients (i.e. ts surviving beyond day 28) will be entered. An acceptable primary graft failure or rejection rate is approximately 10 0/0.

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

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

W0 2013f093919 ions Stable sustained engraftment is defined as neutrophils, more than l for three consecutive days, and platelets, more than 20000/ul for three consecutive days, without transfusion.

Graft rejection is defined as rapid decline of neutrophils, less than 100/pl after documented neutrophil engraftment, with or Without increase of lymphocytes.

Graft failure is defined as failure to reach more than ] neutrophils for three consecutive days and more than 20000/ul 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 able incidence of grade Il-IV acute GVHD is approximately 10 %.

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

Table 1A: Clinical staging of acute GVHD _Rash more than 25 % Bilirubin = 2-3 mg/dl Diarrhea 500-1000 ml _Rash 25-50 % Bilirubin = 3—6 mg/dl Diarrhea 1000-1500 ml _Generalized oderma Bilirubin = 6—15 tug/d1 Diarrhea more than 1500 ml Desquamation and bullae iii/Emu more than 15 Pain or ileus Table 1B: Clinical grading of acute GVHD —_-Gu Functional impairment IV/ life threatening ++ to ++++ ++ to ++++ ++ to ++++ Statistical considerations The time intervals for tment, survival, disease—free survival, relapse rate and risk of transplant-related ity will be calculated from the day of stem cell transplantation. Actuarial curves will be calculated 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, ons in TP53, minus 1 p - n's Lymphoma relapsed after gous transplantation, not eligible for immunotherapy with anti—CD30 - Multiple myeloma relapsing after autologous transplantation, with unfavorable cytogenetics in either partial or complete remission — Severe Aplastic Anemia relapsing after immunotherapy — e 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 (IBMDR) — Presence of haploidentical family donor and a back-up of patient autologous stem cells — Stable clinical conditions and life expectancy of more than 12 weeks ~ Karnofsky ~ more than 70 % — Written informed consent Pre-treatment evaluation - complete clinical history and ation and determination of performance status and body surface area. — complete blood count — blood group, red blood cells subgroups, anti—A and/or anti—B agglutinin - creatinine clearance, uric acid, ferritin, LDH, beta 2 microglobulin, protein electrophoresis, SGOT, SGPT, urine test, blood glucose, blood nitrogen, globulin 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 test. - chest X ray. - lung CT scan, brain CT scan, maxillary sinus CT scan. - dental X ray and examination. — biopsy and bone marrow te for morphologic and cytogenetic analysis, search for a molecular marker (if not known) and FACS analysis (according to underlying disease). - neurologic ation and lumbar puncture in patient at lisk. — radiologic scan (CT, NMR) of the known disease localization. — complete serologic and molecular HLA typing, ML cultures and cytotoxicity test with the ed donors. — cytotoxic anti HLA antibodies.

~ Abdominal echography Exclusion criteria - Patient — History of central nervous system disease localization - vity for HIV, HCV, HCVRNA, HBsAg, HBVDNA — Active and documented pneumonia of any kind, fungal tissue infection, viral positive cultures of respiratory ion or blood - biliiubin of more than 2 times normal — blood creatinine clearance less than 50 mI/min - DLCO less than 50 % of the predicted value — ejection fraction less than 45 % (or dial stroke in the last year) - pregnancy or lactation — psychiatric disorders Eligibility Criteria - Donor — Absence of hematopoietic or marrow function related disease that interferes with the collection of sufficient numbers of normal progenitor cells.

— Absence of any medical condition that would pose a serious health risk by undergoing peripheral blood stem cell harvest — ve HIV, HTLV—l tests — Any healthy family member will be considered for hematopoietic stem cell donation. ion of a donor will be based on typing of HLA—A, B, C, DR loci to be carried out on the recipient, siblings, s and possibly other family members such as aunts, uncles and cousins. A prospective related donor must be at least pically HLA—A, B, C, DR haploidentical to the patient, but can differ for 2-3 HLA alleles on the unshared haplotype.

- Donor will be ed preferentially on the basis of the donor—versus- recipient NK alloreactivity.

Donor Evaluation - Complete history, physical examination and examination of physical veins by the pheresis service for determination of suitability for pheresis via peripheral veins.

- Blood tests: WBC, PLT, Hb, PT, PTT, total protein, albumin, electrolytes, glucose, SGOT/SGPT, alkaline atase, 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 itted disease g must be performed between 30 and 7 days prior to stem cell collection — Chest X—ray — EKG - VNTR analysis by PCR W0 2013(093919 PCT/11.2012/050541 — Donors will be prioritized on the basis of younger 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.

— Presence of any l condition that would pose a serious health risk by undergoing peripheral blood stem cell harvest (i.e. insulin-dependent diabetes, cardiovascular disorders, c atory diseases).

TREATMENT PROCEDURES Mobilization of donor HSC and Graft processing.

Patients are required to have a family donor (aged 18 to 60 yrs), willing and capable of donating filgrastirn/lenogastrim-stimulated peripheral blood hematopoietic cells. Donors will be screened according to Blood Bank general rules. It is advisable to perform an exercise 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. Filgr‘astim/Lenogastrim dosage will be adjusted to maintain white blood cells below 60 x lOg/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 leukapher'esis will be ued 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, tion 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 collect more than 10 x 106 CD34+ cells/kg from an appropriate donor, patients may not proceed 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 (e.g. anti-CD3/l9 ic beads or FACS sorter). Target value of CD34—positive cells will be at least 10 x 106/kg of the recipient body weight .

The apheresis will be performed through the antecubital veins.

Table 2: Conditioning regimen day -6 Fludarabine 3O mg/sqm day -5 Fludarabine 3O mg/sqm day -4 Fludarabine 3O mg/sqin day —3 Fludarabine 3O mg/sqm day —2 TBl 2 Gy single fraction day —1 Rest day 0 Graft day +1 Rest day +2 Rest day +3 CY 50 mg/kg day +4 CY 50 mg/kg As described in Table 2, above, fludarabine will be stered enously daily on 5 sequential days, —7, —6, 5, -4, and —3, at a dose of 30 mg/m2. Each dose will be infused over 30 minutes. 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 through a central access.

CY will be administered enously 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 b. for urate prophylaxis allopurinol 300 mg per os will be given; c. antiemetic therapy will be given ing 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/ uL in absence of fever or bleeding signs; Patient monitoring during treatment a. daily full blood count and differential b. serum creatinine, Na+, K+, Ca++, bilimbin daily during chemotherapy and hyper-hydration c. liver function tests, albumin, ation tests with antitrombin III, cytomegaloviius antigenemia and PCR twice a week. (1. surveillance cultures according to center guidelines TOXICITY EVALUATION Toxicity will be evaluated according to WHO criteria, as indicated in Table 3, below.

Table 3: WHO toxicity criteria “—_--— 2 11.0 g/dl 95-109 g/dl 8.0—9.4 g/dl 23:13 g/dl < 6.5 g/dl Hemoglobin 2 6.8 mmol/l 5.6—6.7 mmol/l 4.9—5.6 inmol/l fl < 4.0 rnmol/l Leukocytes(1000/rmn) 3.0—3.9 2.0—2.9 1.0-1.9 Granulocytes(1000/mm) 1.5—1.9 1.0-1.4 0.5-0.9 Platelets (1000mm) 75-99 25-49 Gross bIOOd Debilitating Hemorrhage None Petechiae Mild blood loss loss blood loss Gastrointestinal ————— $1.25 xN* 126-25 xN* 25 x N* 5.1-10x N* giggimnases (SGOT s 1.25 x N* 12625 x N* 2.6-5 x N* 5110 x N* Alkaline phosphotase S 1.25 X N* 126-25 x N* 2.6—5 X N* 51—10 x N* > 10 x Ni< Erythema, ss/ U106.“ I Oral No change ulcers: can eat . . Alimentation . requires liquid ma not le solids. diet on].

Nausea/vomiting. .

Transient Vomiting Intractable None Nausea . . requiring . . ng vomiting thera.

Diarrhea.

Transient < 2 Tolerable, but > Intolerable, Hemorrhagic None requiring days 2 days dehydration. theray —<125xN* 126-25xN* 2~65xN* 23 + Ne hrotic Proteinuria No change <103 g % 0.3- 1.0 g % >41.0 g % s3’If; 0rme <31 3—1011 >10o/1 uro athy onal Pulmonary No change Mild symptoms Dyspnoea at Complete bed d s-noea rest rest reuuired Fever with drug Fever < 38 °C Fever 38—40 °C Fever > 40 °C oten31on Bronchospasm: $331233?sm Allergic No change Oedema no parenteral thira Anaphylaxis therapy needed .py re uired Exfoliative fiersy uamation dermatitis: MOSt necrosis Cutaneous No change Erythema .q ’ . desquamation, ves1cu1ation, . . requiring . ulceratlon. . pruritus surgical intervention Complete te, Non-reversible ia but patchy alopecia alopecia reversible Maj or infection te Major Infection (specify site) with infection ion . Unifocal PVC, Multifocal cular Rhythm NO change tachycardia, atrial arrhythmi PVC tachycardia > 110 at rest Transient matic Symptomatic Asymptomatic’ symptomatic ction dysfunction Function No change but abnormal dysfunction: no responsive to non-responsive cardiac sign therap re ouire of theray to thera- ade: Asymptomatic Symptomatic: Tamponade : Pericarditis No change surgery effusion no tap required tap required reuired __——_—— Somnolence < Somnolence > State of consciousness Alert 50 % of waking 50 % of Coma therapy Paresthesias Intolerable . and or Perlpheral paresthesias and paresthesias None Paral . ySIS decreased and or marked tendon reflexes weakness motor loss Constipation** None Mild Moderate Abdominal D1stens1on and distensmn vomitinor I_-——-- 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.

Use of narcotics may be helpful in g pain depending on the patient's tolerance.

SUPPORTIVE CARE Monitoring and treatment of bacterial and fungal infections Patients are cared for in isolation rooms with laminar airflow or high-efficiency air—particulate filtration. Liposomal Amphotericin is given at 1 mg/kg/day from day —5 PCT/ILZOIZ/050541 to tment as antifungal prophylaxis. Bacterial infections are monitored by swabs and blood cultures weekly. Intravenous antibiotic therapy is started on the basis of al signs of infection (fever of unknown origin) or positive blood cultures. If the patient is still febrile after 72 hours, empiiic antifungal therapy is statted using either L— AMB 3 mg/kg/day or Von‘conazole 8 day i.v. Vancomycin is added after an additional 72 hours of fever, or in the presence of Gram+ sepsis, or positive blood culture. laxis, 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 infusion. Ganciclovir is reintroduced as preemptive therapy from day +21 until day +360. CMV antigenemia/PCR is determined weekly in blood samples. If CMV antigenemia/PCR develops, patients will be treated with ganciclovir (10 day) or foscarnet (180 mg/kg/day).

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

Post-Transplant Laboratory Evaluation: 1. Daily te ams 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 meiphology is 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 imately 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 phenotypic analysis of circulating lymphocytes, assessment of natural killer and lymphokine activated killer cell function, lymphocyte transformation responses to T—cell and B-cell mitogens and immunoglobulin levels.

W0 20131093919 2012/050541 Follow-up Until day +90 complete blood counts, antigenemia 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 Xray.

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 levels, protein 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 function.

Complete restaging of e will be med 2, 4, 6, 8, 12, 18 and 24 months after transplantation then annually, this will include assessment of donor chimerism by PCR analysis of HLA on peripheral blood and bone marrow cells.

For mance Status grading criteria see Table 4, below.

Table 4: sk Performance scale FUNCTIONAL STATUS RATING GROUP SCORES Normal. No complaints. No evidence of disease.

Able to carry on normal activity. Minor signs or symptoms of disease. 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. es l care and assistance.

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

Requires Very sick. Hospitalisation necessary. 20 institutionalisation Moribund. Fatal processes progressing. 10 (l —39) Dead. 0 MMED INFUSIONS OF DONOR LYMPHOCYTES Donor lymphocyte infusions (DLIs) are effective to treat relapses after allogeneic HSCT. Nevertheless, the success of DLI has been limited to some extent by the morbidity and mortality associated with GVHD. Graded doses of s 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: ]. A recent dose finding study has shown that 1 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): 826, . ts with early molecular and/or 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+ cell/kg. 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 lymphocyte subsets, decreasing their siveness to allogeneic stimuli. The frozen products will be thawed and infused quickly over a period of 5-10 minutes. Patients with acute GVHD or who fail to demonstrate hematological engraftment will not receive any DLI.

Patients with relapsing B cell non-Hodgkin lymphoma will receive rituximab 375 mg/m2 weekly for 4 weeks with DLl 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 post-DLI immunosuppressive agents will be used.

Although the invention has been described in conjunction with c embodiments f, it is evident that many alternatives, modifications and variations will be nt to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, ations and variations that fall within the range and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this cation are herein incorporated in their entirety by into the specification, to the same extent as if WO 93919 each dual 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 constmed as necessaiily limiting.

Claims (40)

WHAT IS CLAIMED IS:

1. 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 wherein said T cell depleted immature hematopoietic cells are obtained in vitro by ting said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an dy to a population of cells comprising the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is d with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a ic field; (iii) washing said matrix to remove unbound cells; and (iv) removing said magnetic field to elute bound cells from said matrix; 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 cture of a medicament for treating a subject in need of a ngeneic cell or tissue graft, wherein said t 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 wherein said T cell ed immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells sing the immature hematopoietic cells that cally binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said re hematopoietic cells cally bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) washing said matrix to remove unbound cells; and (iv) removing said magnetic field to elute bound cells from said matrix; 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 ing transplantation of a cell or tissue graft, in the manufacture of a ment 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 d intensity conditioning protocol.

4. The use of claim 1 or 2, wherein the medicament is adapted for administration with an in—vivo T cell debulking 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

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 hematopoietic cells are selected from the group consisting of T cell depleted bone marrow cells, T cell ed G-CSF mobilized peripheral blood progenitor cells, T cell depleted cord blood, d CD34+ cells attained by ve selection from bone marrow and/or from G—CSF mobilized peripheral blood progenitor cells, and ex—Vivo expanded CD34+ cells.

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

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

10. The use of claim 1 or 2, n when said surface marker is a T cell surface marker, said unbound cells comprise T cell depleted immature hematopoietic cells.

11. The use of claim 10, wherein said T cell surface marker is selected from the group consisting of CD2, CD3, CD4, CD8 and TCRoz/B.

12. The use of claim 1 or 2, wherein when said surface marker is an immature poietic cell surface marker, said bound cells comprise T cell depleted immature hematopoietic cells.

13. The use of claim 12, wherein said re hematopoietic cell surface marker is selected from the group consisting of CD34, CD33 and CD131.

14. The use of claim 1 or 2, further comprising separating B cells from said T cell depleted immature hematopoietic cells by the use of an antibody that specifically binds to a B cell surface marker.

15. The use of claim 14, wherein said B cell surface marker is selected from the group consisting of CD19 and CD20.

16. The use of claim 1 or 2, wherein said matrix is a ferromagnetic matrix.

17. The use of claim 1 or 2, wherein said matrix ses spheres of magnetically susceptible or ferromagnetic material.

18. The use of claim 1 or 2, wherein said magnetically responsive agent comprises a superparamagnetic particle.

19. The use of claim 18, wherein said superparamagnetic particle is conjugated to said antibody in combination with an anti-immunoglobulin, an avidin and or anti- hapten—specific microbead.

20. The use of any one of claims 1, 2 or 18, wherein said separating said T cells from said immature poietic cells is effected using a high gradient magnetic separation (HGMS).

21. The use of claim 1 or 2, wherein said separating said T cells from said immature hematopoietic cells is ed using a separation column.

22. The use of claim 1 or 2, wherein said antibody is selected from the group consisting of an anti—CD8 antibody, an anti—CD4 dy, an D3 antibody, an anti- CD2 antibody, an anti-TCRoz/B antibody, an anti—CD19 dy, and an anti-CD20 antibody, an anti-CD21 antibody an anti-CD34 antibody, an anti-CD33 antibody and an anti-CD131 antibody.

23. The use of claim 1 or 2, wherein said T cell depleted re hematopoietic cells are from a non-syngeneic donor.

24. The use of claim 23, wherein said non—syngeneic donor is allogeneic or xenogeneic with respect to said subject.

25. The use of claim 24, wherein said allogeneic donor is selected from the group consisting of an HLA matched sibling, an HLA d unrelated donor, an HLA haploidentical related donor and a donor displaying one or more disparate HLA determinants.

26. The use of claim 1 or 2, wherein said subject is a human subject.

27. The use of claim 4, wherein said in-vivo T cell debulking is ed by antibodies.

28. The use of claim 27, wherein said antibodies comprise at least one of an anti—CD8 antibody, an anti-CD4 antibody, an anti~thymocyte globulin (ATG) dy, an anti-CD52 antibody and an anti-CD3 (OKT3) antibody.

29. The use of claim 3, wherein said reduced intensity conditioning comprises a non-myeloablative ioning protocol.

30. The use of claim 29, wherein said non—myeloablative conditioning ses at least one of a total body irradiation (TBI), a total lymphoid irradiation (TLI), a chemotherapeutic agent and/or an antibody immunotherapy.

31. The use of claim 30, wherein said TBI comprises a single or onated irradiation dose within the range selected from the group ting of 1—7.5 Gy and 1—3.5

32. The use of claim 30, wherein said chemotherapeutic agent comprises at least one of Busulfan, Fludarabine, Melphalan and Thiotepa.

33. The use of claim 30, wherein said antibody comprises at least one of an anti-CD52 dy, an anti-thymocyte globulin (ATG) antibody and anti-CD3 (OKT3) antibody.

34. 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 wherein said T cell depleted re hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that specifically binds to a surface marker, n said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells sing said immature hematopoietic cells specifically bound to said dy labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) washing said matrix to remove unbound cells; and (iv) removing said magnetic field to elute bound cells from said matrix; and a therapeutically effective amount of cyclophosphamide, n said therapeutically effective amount ses 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, wherein said subject has a ant disease.

35. Use of a dose of T cell depleted immature hematopoietic cells, n said T cell depleted immature poietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose ses at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically sive agent; (ii) immobilizing said population of cells comprising said re hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field; (iii) g said matrix to remove unbound cells; and (iv) removing said magnetic field to elute bound cells from said matrix; 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 stration to the subject following transplantation of immature hematopoietic cells, in the manufacture of a medicament for ng a subject in need of an immature hematopoietic cell lantation, n said subject has a non-malignant disease.

36. Use of a dose of T cell depleted immature hematopoietic cells from a non- syngeneic donor, 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 n said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells comprising said immature hematopoietic cells specifically bound to said antibody d with said magnetically responsive agent in a matrix through a magnetic field; (iii) washing said matrix to remove unbound cells; and (iv) ng said magnetic field to elute bound cells from said matrix; and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg per kilogram body weight of the t, and wherein said cyclophosphamide is for stration to the subject following transplantation of a cell or tissue graft, in the manufacture of a medicament for inducing donor c tolerance in a subject in need of a non—syngeneic cell or tissue graft, n said subject has a malignant disease.

37. Use of a dose of T cell depleted immature hematopoietic cells from a non- syngeneic donor, wherein said T cell depleted immature 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 wherein said T cell depleted immature hematopoietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells, by a method comprising: (i) adding an antibody to a population of cells comprising the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent; (ii) immobilizing said population of cells sing said immature hematopoietic cells cally bound to said dy labeled with said magnetically responsive agent in a matrix through a ic field; (iii) washing said matrix to remove unbound cells; and (iv) removing said magnetic field to elute bound cells from said matrix; 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 lantation of a cell or tissue graft, in the cture of a medicament for inducing donor specific tolerance in a subject in need of a non—syngeneic cell or tissue graft, wherein said subject has a non-malignant disease.

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 wherein separating said T cells from said re hematopoietic cells is effected in vitro based on a product secreted by said T cells; 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.

39. 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 n separating said T cells from said immature hematopoietic cells is effected in vitro based on a product secreted by said T cells; and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 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 ment for treating a subject in need of a non—syngeneic cell or tissue graft, wherein said subject has a non—malignant e.

40. Use of a dose of T cell depleted immature hematopoietic cells, wherein said T cell ed immature hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and n said dose comprises at least about 5 X 106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell ed immature poietic cells are obtained in vitro by separating said T cells from said immature hematopoietic cells based on an expression of at least one surface marker of T cells selected from the group consisting of CD2, CD3, CD4, CD8, TCR