Use of IL15 Stimulated, CD3/19 Depleted Transplants From Haploidentical Donors In Pediatric Malignancies
Authors: Lang Peter , Pfeiffer Matthias , Teltschik Heiko-Manuel , Feuchtinger Tobias F. , Ebinger Martin , Mueller Ingo , Schumm Michael , Handgretinger Rupert 
Published: October 2019

Abstract
Abstract 3548
T and B cell depleted haploidentical grafts and a melphalan based intensity reduced regimen result in low toxicity and stable event free survival in patients with leukemias in CR1-3. However, patients with active disease or with second or subsequent transplantation show unacceptable relapse rates. In an ongoing study, 36 pediatric patients with acute leukemias and advanced MDS (median age: 11 years) received melphalan (2×70mg/m2), fludarabine (4×40mg/m2) or clofarabine (4×50mg/m2), thiotepa (10mg/kg) and OKT3 (0.1mg/kg). T and B cells were depleted by antiCD3/antiCD19 coated magnetic microbeads, whereas NK cells remained into the grafts (median number= 120×106/kg).
Remission status was: CR1-3=18, NR=18, 18/36 already received previous allogeneic transplantations. Relapse rates at 2 years were 20% (CR patients) and 73% (active disease patients or 2nd trp). Thus, we investigated options to reduce the risk of relapse by increasing antileukemic activity of donor NK cells in the grafts in vitro. Over night incubation with Interleukin 15 increased NK activity most effectively (specific lysis at E:T=20:1 against K562: 28% prior to and 71% after stimulation, n=10). After additional IL2 stimulation a 22 fold increase in thymidine uptake indicated proliferation of NK cells (n=5). Due to the profound depletion, no T cell proliferation was detectable. Based on these results, we started a pilot study with ex vivo IL15 stimulated grafts in 4 patients at very high risk (ALL, 3rd relapse, active disease (n=1); ALL 2nd relapse, remission (n=1); AML 1st relapse, active disease (n=2)). All patients received a backbone of unstimulated cells at day 0 to ensure engraftment. Additionally, parts of the grafts were incubated over night, washed four times and afterwards infused at day +1 (median numbers: CD56+CD3- =9.4×106/kg (range 3.7–24.4); CD34=1.5×106/kg; CD14=34×106/kg, CD3=0.01×106/kg). No acute side effects occurred. All patients engrafted within 12 days. 3 patients had acute GvDH grade 0-I, 1 patient had GvHD grade III. Recovery of NK cells was remarkably fast (526 CD56+/μl at day 14 posttransplant versus 256 CD56+/μl in patients without IL15 stimulation (means)). After additional administration of IL2 in vivo (1×106 Units/m2/day s.c.) high NK activity (specific lysis>90% against K 562, E:T=20:1) was detectable in peripheral blood. Two patients are disease free (day 154 and 674 posttransplant), 2 patients died from relapse (day 56 and 64).

Conclusions:
ex vivo stimulation with IL15 strongly increases cytotoxic activity of NK cells in T and B cell depleted grafts from haploidentical donors and can counterbalance G-CSF mediated inhibitory effects. Those grafts were infused without any acute side effects and resulted in a fast recovery of functional donor NK cells. Potential interactions with stem cells and stem cell derived NK reconstitution have to be investigated. Further studies have to evaluate if this approach might contribute to reduce relapse rates in high risk patients.

Disclosures:
No relevant conflicts of interest to declare.