Genetically engineered natural killer (NK) cell immunotherapy for children poor risk b-cell (CD20+) leukemia and lymphoma (L/L).

Abstract

e13022 Background: A majority of children with CD20+ L/L at relapse have a chemotherapy resistant phenotype (Cairo et al Blood, 2007; JCO, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this poor risk population. NK cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Beziat V et al, Leukemia, 2009) but cell number and tumor recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). PBNK cells expanded with genetically engineered K562-mbIL15-41BBL cells (geK562) have been previously reported (Imai C et al, Blood. 2005). Objective: We investigated the functional activities and cytolytic effect of anti-CD20 chimeric antigen receptor (CAR+) engineered PBNK cells expanded with mK562 against CD20+ L/L both in vitro and in vivo. Methods: Peripheral blood mononuclear cells (PBMC) were expanded with mitomycin C treated geK562 cells in culture medium with 10 IU/ml IL-2 for 7 or 14 days. CD56 and CD3 expression were evaluated by flow cytometry. Retrovirus preps that express CAR+ or CAR- were generated independently. The CAR+ was constructed in a MSCV-anti-CD20BB-CD3-zeta-GFP plasmid (generously supplied by Dario Campana, MD, PhD). Expanded PBMC were transduced with retroviruses as described (Imai C et al, Blood. 2005). NK cytotoxicity was assessed by europium release assay at 2:1 E:T ratio against CD20+ Ramos. Results: CD56+CD3- PBNK cells were significantly increased compared to media alone at day7 (60.94+ 3.63% vs 8.05+0.49%, n=6, p<0.001). CD56-CD3+ PBT cells were significantly reduced compared to media alone at day 7 (22.08+2.22% vs 75.73+0.75%, n=6, p<0.001). CAR+ and CAR- retrovirus supernants infected expanded PBMC at 1%-10% range. The anti-CD20 CAR expression was further confirmed by flow cytometry and western blot. We also observe that cytotoxicity was enhanced with CAR+ PBNK compared to CAR- PBNK (41+ 1.1% vs 24.5+ 3.7%) against Ramos at E:T ratio 2:1. Conclusions: PBNK can be expanded with geK562. Anti-CD20 CAR enhances PBNK anti-tumor activity against CD20+ Ramos. Future directions include characterizing the cytotoxicity activity of engineered PBNK against L/L in vitro and survival in xenogafted mice.