Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation

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.