Potential and limitations of HSV-TK-transduced donor peripheral blood lymphocytes after allo-BMT

In allogeneic marrow transplantation (BMT), donor lymphocytes play a central therapeutic role in both graftversus-leukemia and immune reconstitution. However, their use is limited by the risk of severe graft-vs-host disease (GvHD). In a pilot study, patients who relapsed or developed an Epstein-Barr virus (EBV)-induced lymphoma after BMT were treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-tk) suicide gene. The transduced lymphocytes survived for over 12 months, in high proportions (up to 13.4% of circulating PBLs) resulting in anti-tumor activity in over 50% of patients. Three patients developed GvHD, which could be controlled by ganciclovir-induced elimination of the transduced cells [Bonini C, et al, (1997) Science 276: 1719]. This pilot study is now extended into international multicenter collaborative clinical trials. These new studies are now designed taking into account the potential limitations detected in the pilot study and described here. Three patients in this series could not completely benefit from the strategy based on the HSV-tk-engineered donor lymphocytes. Two patients developed a specific immune response against the HSV-tk transduced cells resulting in immediate elimination of genetically engineered cells. One patient, who developed chronic GvHD after achieving complete remission of leukemic relapse, showed partial resistance to ganciclovir-mediated elimination of transduced cells. In order to evaluate the in vivo immune response against the product of the transgenes, we tested the ex vivo cytotoxic response of the circulating PBLs against donor lymphocytes independently transduced with the different genes of the SFCMM-2 retroviral vector. Specific immune response was observed against the cell surface marker (delta LNGFR). This phenomenon was observed in two patients, who received the first infusion of genetically modified donor cells late after BMT (8 and 9 months), documenting the role of the recipient immune reconstitution. In order to characterize the partial ganciclovir resistance observed in chronic GvHD, circulating PBL obtained from the patient before and after ganciclovir treatment were analysed ex vivo; No difference in ganciclovir sensitivity was observed between transduced T-cells obtained ex vivo before and after in vivo ganciclovir treatment. Also no difference was observed in the co-expression of delta LNGFR and CD3, CD4, CD8, CD56. Ex vivo selection of transduced PBL resulted in a polyclonal cell population, as documented by Southern blot. All the results obtained documented the integrity of the integrated transgenes as well as their high level of expression, and suggest that the ganciclovir resistance of chronic GvHD is mainly related to the cell-cycle dependence of the HSV-TK/ganciclovir treatment. In an attempt to circumvent this limitation, a retroviral vector with a 2 log higher ganciclovir sensitivity (LD 50 = 0,1 muM) has been developed. Additionally, in order to reduce the immunogenicity of transduced cells, the NEO gene has been removed from the new vector. This new vector named SFCMM-3 is now utilized in prophylactic protocols that take into account the immunogenic potential of transduced cells