ALBERT

Description: T-cell depletion (TCD) of allogeneic stem cells transplantation (HSCT) is the most powerful approach to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. However, the prolonged immune deficiency resulting from TCD significantly impairs the outcome of HSCT from a haploidentical family donor (haplo-HSCT). In a phase I-II clinical trial (protocol MMTK007), we showed that the add-back of suicide-gene transduced donor lymphocytes (TK+ cells) provides early immune-reconstitution (IR), crucial to avoid opportunistic infections and disease relapse. While the achievement of an early and sustained IR significantly reduced the incidence of infectious complications and mortality, the kinetic of abatement of viral reactivations differed in different patients, suggesting variability in the repertoire, function and persistence of viral-specific T cells. To gain insights on the activity of the post-transplant immune system, we focused on immune responses to cytomegalovirus (CMV) and Ebstein Barr virus (EBV) as clinical paradigm of an effective immune system. In our trial 29 patients (median age 52y) were transplanted for high-risk leukemia. Seventeen pts received TK-DLI starting at day +42, 14 pts obtained prompt immune reconstitution with CD3+ 〉100/mcl at day +86 (median) from SCT and day +21 from TK-DLI. Twelve/14 experienced CMV reactivation, 3 reactivation/patient (median), each reactivation required 14 days (median) of pre-emptive treatment with foscarnet to achieve a complete clearance; in 2/38 events, a CMV antigenemia persistent during foscarnet treatment was cleared by ganciclovir administration. In this series, the last CMV reactivation requiring pre-emptive treatment happened at day +84 from SCT and day +19 from TK-DLI. Nine/14 experienced EBV reactivation, 1 reactivation/patient (median), 6 patients required treatment with rituximab (375 mg/mq weekly) for 3 cycles (median). In 3 cases we documented EBV lymphoproliferative disease, that were completely controlled with the treatment. The proportion of lymphocytes committed to produce IFN-γ upon CMV or EBV stimulation normalized within the first 3 months post transplant. The analysis of CMV and EBV T-cell response showed a prevalent host-restriction pattern, suggesting active modulation of the T-cell repertoire by the host environment. The TCR-Vβ repertoire progressively changed from oligoclonal into polyclonal and normalized by 1 year after transplant. The progressive acquirement of a protective, host-restricted, anti-viral response, highly correlated with a decline in the occurrence of any infectious event, that were nearly abrogated by 6th month post transplant. The overall cumulative infectious mortality beyond day +100 post transplant was 12,5% in TK treated patients demonstrating the efficacy of this strategy in building a protective immune system.

Description: The outcome of haplo-SCT is limited by delayed immune reconstitution resulting in a high rate of late mortality and relapse. Here, we report results of a phase II multicenter trial (MM TK007) of early add-backs of donor lymphocytes genetically engineered to express the herpes simplex thymidine kinase (TK) suicide gene after haplo-SCT in inducing immune reconstitution and selective control of GvHD by ganciclovir. Twentysix advanced age pts (median age 51, 17–63) were transplanted for high risk leukemia; disease status at SCT was CR1 (8), CR2 (7), refractory (11). A median of 12.2x106/kg (7.3–16.8) CD34+ selected (Clinimacs) and 1x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning. 24/26 pts engrafted with a median time of 14 d (8–21) for ANC 〉1.0x109/l and 13 d (11–24) for plt 〉50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-add-back. Sixteen pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42 and 13 pts obtained CD3+ 〉100/mcl at a median time of 91 d (61–127) from SCT and 24 d (14–42) from TK-DLI. Transduced cells were documented ex vivo in all pts and represented a median of 48% (10–90) of CD3+ cells. Five pts developed acute GvHD, (grade I to IV) that was always completely abrogated by ganciclovir. In patients in CR at time of SCT who were alive at d +42 and received add-backs of Tk cells, OS rate was 46% at 800 days (intention-to-treat analysis: 38% OS at 800 days post-SCT). Of significance, the cumulative incidence of TRM and relapse showed a 40% probability of mortality with a median time of death of 90 days and last event at day +166. This figure indicate that TK cell add-backs abolish late mortality after CD34+ SCT in adults. In patients in relapse at time of HSCT, a median OS of 201 days was obtained in ITT, with a significant advantage on expected survival without transplantation (60 d) and superior results as compared to haplo EBMT registry (80 d). The 2-year estimation of events of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from late infectious mortality after haplo-SCT. We believe that these results are due to the rapid development of a wide T cell repertoire obtained by TK cell infusions. Immunological follow-up showed Th1/Tc1 effector memory T cells, with a wide TCR repertoire in the first 3 months after SCT in all patients. High frequencies of T cells specific for CMV (median: 35 and 93 spots/105 cells with CMV-infected donor and host fibroblasts) and EBV (median: 58 and 41 spots/105 cells with donor and host EBV-LCL) were detected by gIFN ELISpot at time of immunereconstitution, and correlated with complete control of viral infections. Normalization of the T cell repertoire was documented by spectratype, immune-phenotype for naïve and memory T cell subsets and gIFN ELIspot 6 months after treatment. A phase III randomized multicentric study will start in 2006.