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The infusion of donor lymphocytes after allogeneic bone; marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse (1-7), and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Barr virus-induced lymphoproliferative disorders (EBV-BLPD) (8) or reactivation of CMV infection (9). Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GVHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor anti-tumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors (10,11). This protocol has been designed to evaluate in the contest of allogeneic BMT: 1-the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2--the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3-the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes. Patients will be enrolled in three groups: A-patients in complete remission after a T depleted allo-BMT, in order to prevent disease relapse; B-patients with relapse of hematologic malignancies after allo-BMT, in order to achieve a GvL effect; C-patients with an EBV-BLPD after allo-BMT, in order to provide donor EBV-specific T-cells. For this purpose, donor peripheral blood lymphocytes (PBL), will be transduced with a suicide retroviral vector containing two genes: the first coding for the thymidine kinase of the herpes simplex virus (HSV-tk) and the second coding for the low affinity receptor for NGF(12) truncated of its intracellular domain (Delta NGFR). The HSV-tk confers ganciclovir sensitivity (13), thus allowing in vivo selective downregulation of all transduced allogeneic cells in case of severe GvHD. The Delta NGFR will be used as a cell surface marker allowing rapid in vitro immunoselection of transduced cells, and ex vivo detection and characterization of the transduced cells after infusion. The infusion of donor lymphocytes after allogeneic bone marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse, and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Parr virus-induced lymphoproliferative disorders (EBV-BLPD) or reactivation of CMV infection. Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GvHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor anti-tumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors. This protocol has been designed to evaluate in the contest of allogeneic BMT: 1 - the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2 - the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3 - the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes. Patients will be enrolled in three groups: A-patients in complete remission after a T depleted allo-BMT, in order to prevent disease relapse; B - patients with relapse of hematologic malignancies after allo-BMT, in order to achieve a GvL effect; C - patients with an EBV-BLPD after allo-BMT, in order to provide donor EBV-specific T-cells. For this purpose, donor peripheral blood lymphocytes (PBL), will be transduced with a suicide retroviral vector containing two genes: the first coding for the thymidine kinase of the herpes simplex virus (HSV-tk) and the second coding for the low affinity receptor for NGF truncated of its intracellular domain (ΔNGFR). The HSV-tk confers ganciclovir sensitivity, thus allowing in vivo selective downregulation of all transduced allogeneic cells in case of severe GvHD. The ΔNGFR will be used as a cell surface marker allowing rapid in vitro immunoselection of transduced cells, and ex vivo detection and characterization of the transduced cells after infusion.

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The extensive exploitation of the antitumor effect of donor lymphocytes infused after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is limited by the risk of graft-versus-host disease (GvHD). To overcome this limitation, we investigated the therapeutic potential of donor lymphocytes engineered with the suicide gene thymidine kinase of herpes simplex virus (TK) in 23 patients experiencing recurrence of hematologic malignancies after allo-HSCT. Long-term follow-up of infused patients included analysis of engraftment of genetically engineered lymphocytes, in vivo assessment of antitumor effect, and control of GvHD by ganciclovir. All 17 patients evaluable for engraftment and graft-versus-leukemia (GvL) had circulating TK+ cells detectable beginning at a median time of 18 days. Eleven patients (65%) experienced a substantial clinical benefit resulting in 6 (35%) complete remissions and 5 (29%) partial responses. The antitumor effect tightly correlated with the in vivo received ganciclovir, resulting in elimination of TK+ cells and effective and selective treatment of GvHD. Immunization against HSV-TK was observed in 7 patients but did not preclude an effective GvL. These data validate the feasibility, safety, and efficacy of TK+ cells in the context of allografting and represent the basis for a broader application of this technology. The extensive exploitation of the antitumor effect of donor lymphocytes infused after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is limited by the risk of graft-versus-host disease (GvHD). To overcome this limitation, we investigated the therapeutic potential of donor lymphocytes engineered with the suicide gene thymidine kinase of herpes simplex virus (TK) in 23 patients experiencing recurrence of hematologic malignancies after allo-HSCT. Long-term follow-up of infused patients included analysis of engraftment of genetically engineered lymphocytes, in vivo assessment of antitumor effect, and control of GvHD by ganciclovir. All 17 patients evaluable for engraftment and graft-versus-leukemia (GvL) had circulating TK+ cells detectable beginning at a median time of 18 days. Eleven patients (65%) experienced a substantial clinical benefit resulting in 6 (35%) complete remissions and 5 (29%) partial responses. The antitumor effect tightly correlated with the in vivo received ganciclovir, resulting in elimination of TK+ cells and effective and selective treatment of GvHD. Immunization against HSV-TK was observed in 7 patients but did not preclude an effective GvL. These data validate the feasibility, safety, and efficacy of TK+ cells in the context of allografting and represent the basis for a broader application of this technology.

Hematopoietic SCT (HSCT) from HLA haploidentical family donors is a promising therapy for high-risk hematological malignancies. In the past 15 years at San Raffaele Scientific Institute, we investigated several transplant platforms and post transplant cellular-based interventions. We showed that T cell-depleted haploidentical transplantation followed by the infusion of genetically modified donor T cells (TK007 study, Eudract-2005-003587-34) promotes fast and wide immune reconstitution and GvHD control. This approach is currently tested in a phase III multicenter randomized trial (TK008 study, NCT00914628). We targeted patients with advanced leukemia with a sirolimus-based, calcineurin inhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated PBSCs from haploidentical family donors (TrRaMM study, Eudract 2007-5477-54). Results of these approaches are summarized and discussed. Hematopoietic SCT (HSCT) from HLA haploidentical family donors is a promising therapy for high-risk hematological malignancies. In the past 15 years at San Raffaele Scientific Institute, we investigated several transplant platforms and post transplant cellular based interventions. We showed that T cell-depleted haploidentical transplantation followed by the infusion of genetically modified donor T cells (TK007 study, Eudract-2005-003587-34) promotes fast and wide immune reconstitution and GvHD control. This approach is currently tested in a phase III multicenter randomized trial (TK008 study, NCT00914628). We targeted patients with advanced leukemia with a sirolimus-based, calcineurin inhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated PBSCs from haploidentical family donors (TrRaMM study, Eudract 2007-5477-54). Results of these approaches are summarized and discussed.

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSC transplantation (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10 anergized donor T cells (IL-10 DLI) contained T regulatory type 1 (Tr1) cell specific for the, host alloantigens, limiting donor-vs-host reactivity, and memory T cells able to respond to pathogens. IL-10 DLI were infused in 12 patients with the goal of improving immune-reconstitution after haplo-HSCT without increasing the risk of GvHD. IL-10 DLI led to fast immune-reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10 DLI. These four patients are alive, in complete disease remission and immune suppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1- specific biomarkers in vivo. Gene expression profiles of immune-reconstituted patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves the way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine produced by stromal cells that acts as a chemoattractant for human CD34+ progenitor cells. We investigated the expression of CXCR4, the receptor for SDF-1, on CD34+ cells from different hematopoietic sites and developmental stages. CXCR4 was detected by flow cytometry on 37 % of fetal bone marrow (BM) [gestation weeks (gw) 14-23] and 40% of adult BM CD34+ cells. Interestingly, in fetal liver CD34+ cells, CXCR4 was expressed at lower levels at later stages (9%, gw 20-23) compared to early stages of development (39%, gw 7.5-18), suggesting a development-related change in the migratory capacity of progenitors. CXCR4 was detected at similar levels on both phenotypically primitive and committed progenitors from fetal and adult sites. However, B cell lineage progenitor and precursor cells expressed CXCR4 at the highest density (80% of BM CD34+/CD10+ pro-B cells are CXCR4+). CXCR4 was also expressed in the fetal thymus in early T cell precursors and found to be down-regulated during T cell maturation. Finally, we found that stem cell factor, alone or in combination with other cytokines, can up-modulate CXCR4 expression on CD34+ cells by three- to fourfold. In conclusion, our results suggest that CXCR4 may play an important role in the local and systemic trafficking of human CD34+ cells as well as in human B lymphopoiesis and that its expression can be modulated by cytokines.

In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GVHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GVHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients. In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GVHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GVHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients.