ALBERT

Description: Abstract 513 UCB transplantation (UCBT) is associated with greater risk of graft rejection and, in particular in double UCBT, with graft vs. host disease (GVHD). Our group has shown in mouse models that ex vivo activated and expanded Tregs improve engraftment and reduce and treat GVHD. We here report on the first clinical trial to study the safety of the infusion of UCB Tregs in patients receiving a nonmyeloablative double UCB transplant. We enrolled 19 patients (pts) (age 25-65 yrs; weight 52-133; male 9; CMV seropositive 6) with high risk or advanced leukemia (n=12) and lymphoma/CLL (n=7). All patients received the same nonmyeloablative conditioning consisting of cyclophosphamide 50mg/kg/×1day, fludarabine 40 mg/m2/×5d, and total body irradiation 200 cGy/×1d; immunosuppression was cyclosporine A/mycophenolate mofetil (MMF) in the first 17 and sirolimus (Siro)/MMF in the last 2 pts. Tregs were obtained from a 3rd UCB unit and after CD25+ selection (Miltenyi CliniMACS) were activated and expanded in the presence of anti-CD3/CD28 mAb coated beads and IL-2 for 18±1 days. Treg dose escalation levels were 1, 3, 10, 30 × 105/kg on day +1, and 30 × 105/kg on days +1 and +15 after UCBT. After CD25+ selection the median % CD4+/CD25+ was 62% (range , 11-87%) and following culture was 86% (r, 62-97%). Median fold expansion was 198 (r, 13-1169); median mixed lymphocyte reaction suppression at a 1:4 ratio post-culture was 86% (39-95%). All products achieved lot release. Pts were monitored for 48h post-infusion and no dose limiting or serious adverse event was observed. After infusion an increase in the proportion of peripheral blood comprised of CD4+FoxP3+CD127- cells was observed. Donor Tregs were clearly detected in all pts receiving Tregs that were HLA disparate with the pt and graft donor units (Figure 1). Mixed day 21 donor graft UCB chimerism was observed in 59% (10/19) of pts vs. 35% (38/107) in historical controls. The cumulative incidence of grades II-IV acute GVHD was 47% (95%CI, 24-71) and III-IV 16% (95%CI, 0-33). In pts receiving ≥ 30 × 105/kg (n=14), grades II-IV acute GVHD was 43% (95%CI, 16-70) and III-IV 7% (95%CI, 0-21). Compared to historical controls receiving the same conditioning regimen, the median time to the development of acute GVHD was longer (Figures 2), although not statistically significant. Neutrophil recovery was 95% (95%CI, 79-100) at median of 8.5 days (r, 3-36), platelet recovery 〉50,000/mL was achieved in 74% (95%CI, 53-95) at a median 43 days (r, 27-57), cumulative incidence of opportunistic infections (fungal + viral) at 100 days was 32% (95%CI, 11-53), treatment related mortality at 100 days was 11% (95%CI, 0-25), and disease free-survival was 40% (95%CI, 15-65%). None differed from historical controls. Based on our results, the co-infusion of ex vivo expanded and activated UCB-derived Treg to recipients of nonmyeloablative UCBT 1) is safe at the tested dose levels, 2) leads to detectable increased in Treg-donor unit derived circulating CD4+FoxP3+CD127- cells, and 3) results in an increased the proportion of mixed chimerism at day +21. Identification of the Treg MTD in the context of Siro/MMF immunosuppression will set the stage for the planned definitive studies that will establish the true impact of Tregs on engraftment and GVHD and for future studies in the treatment of autoimmune disease. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3036 Poster Board II-1012 The potential role of allogeneic natural killer (NK) cells for therapy of refractory lymphoma is supported by the curative potential of allogeneic transplantation for lymphoid malignancies. Haploidentical donor derived NK cells may overcome Class I MHC Ag mediated inhibition and deliver an NK versus lymphoma effect. In a Phase II study we evaluated allogeneic NK cell infusions with Rituximab and IL-2 in a non-transplant setting to determine the expansion of NK cells in vivo and the clinical response in patients with refractory B-cell non-Hodgkin lymphoma (NHL). Six patients with advanced NHL received conditioning with Rituximab 375mg/m2 days -8,-1,+6,+15; Cyclophosphamide 60 mg/kg IV day -5; Fludarabine 25 mg/m2 IV days -6 through -2 as immunosupression to permit homeostatic expansion of allogeneic donor NK cells. Peripheral blood cells were obtained by lymphapheresis from unmobilized, HLA-haploidentical donors and selected for “killer immunoglobulin receptor” (KIR) ligand mismatch when available (3 out of 6 patients). Donor peripheral blood cells were enriched for NK cells with the Miltenyi CliniMACS device by depletion of T (CD3+) cells. The donor NK cells were then activated by overnight incubation with IL-2 (1,000 U/mL) and infused at a median nucleated cell dose of 2.27 ±0.4 × 107/kg. Subcutaneous IL-2 10×106 units (qod x 6 doses) was given to facilitate NK cell survival and expansion. All patients were evaluable for toxicity and efficacy. Patients tolerated the NK infusion well with only transient grade 1-2 toxicity and 5 received all 6 scheduled doses of IL-2. IL-2 activated donor NK cell products showed 〉 55% cytotoxicity against K562 targets. After IL-2 therapy, we observed a median absolute lymphocyte count of 980 ±440/μL. All cells were of recipient origin with no detectable donor NK cells. Importantly, in all patients the median number of host regulatory T cells (T regs phenotype CD4+Foxp3+CD127−) post treatment was significantly increased compared to pre-treatment (day 14 T regs: 134 ±141 cells/μL versus pre-treatment T regs: 24 ±12 cells/μL; P=0.06). To investigate the possibility of NK trafficking to affected lymph nodes, we performed fine needle aspiration of palpable tumor in 1 patient and demonstrated a low level of donor DNA by RFLP testing (2.5% donor chimerism). Simultaneous absence of NK cells in peripheral blood in the same patient suggested NK cell tissue homing to lymphoma-bearing nodes. Three patients achieved a partial remission (PR), one of whom proceeded to non-myeloablative cord blood allograft 2 month after NK cell infusion; two remain in partial remission after 1 and 4 months of follow-up. The trial failed to achieve prospective statistical parameters established to detect circulating NK cell expansion rate and will be modified. Conclusions This “proof of principle” study demonstrated lack of in vivo expansion of haploidentical NK cells in peripheral blood of patients with lymphoma. However, we identified host factors that interfered with NK cell expansion, including T reg proliferation and possibly inadequate immunosupression, and additionally, the finding of donor DNA in sites of tumor suggested donor NK cell localization to extravascular or tumor sites. Novel approaches to adoptive NK cell therapy trials should incorporate strategies to eliminate or prevent T reg expansion using alternate lymphodepleting regimens. Disclosures No relevant conflicts of interest to declare.