Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia
Authors: Schlegel Patrick , Klose Chihab , Kyzirakos Christina , Seidel Ursula J.E. , Witte Kai , Horrer Annika , Matela Marie , Pfeiffer Matthias , Handgretinger Rupert , Lang Peter 
Published: October 2019

Abstract
GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded &gt;1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia.

Disclosures
No relevant conflicts of interest to declare.