Background: Patients with CD20+ L/L at relapse have a chemotherapy resistant phenotype and a dismal prognosis (Cairo et al, Blood, 2007). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (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). Ex vivo expansion of PBNK cells with genetically engineered K562-mbIL15-41BBL cells (modified K562) and the expanded PBNK cells transduced with chimeric anti-CD19 antigen receptor (CAR) have been previously reported (Imai C et al, Blood. 2005). Objective: We investigated the functional activities and cytolytic effect of chimeric anti-CD20 antigen receptor (CAR+) engineered PBNK cells expanded with K562-mbIL15-41BBL cells (modified K562) against CD20+ L/L both in vitro and in vivo. Methods: Whole peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. PBMC were incubated with mitomycin C modified K562 cells in culture medium with 10 IU/ml IL-2 for 7 or 14 days. CD56 and CD3 expression were evaluated by flow cytometry. The CAR+ was constructed in a MSCV-anti-CD20BB-CD3-zeta-GFP plasmid (generously supplied by Dario Campana, MD, PhD, St. Jude Children's Hospital). CAR+ and CAR retroviruses were generated in 293T cells with helper plasmids. Jurkat cells were used to examine infection efficiency. Anti-CD20 CAR expression was examined by flow cytometry and by western blot of whole cell lysates for CD3zeta. PBMC stimulated with modified K562 were transduced with retroviruses as previous described (Imai C et al, Blood. 2005). PBNK cells cytotoxicity was assessed by europium release assay at 2:1 E:T ratio against CD20+ Ramos. Results: CD56+CD3 PBNK cells were expanded significantly more following modified K562 stimulation compared to media alone at day7 (60.94+ 3.63% vs 8.05+0.49%, n=6, P<0.001). CD56CD3+ PBT cells were significantly reduced compared to media alone at day7 (22.08+2.22% vs 75.73+0.75%, n=6, P<0.001). CAR+ and CAR retrovirus supernants infected Jurkat cells at 40%-97% efficiency and infected expanded PBMC at 1%-10% range (n=3). The anti-CD20 CAR expression was further confirmed by flow cytometry and western blot. We also observe that cytotoxicity was enhanced with anti-CD20 CAR+ PBNK compared to CAR PBNK (41+ 1.1% vs 24.5+ 3.7%, n=3) against Ramos at effector to target ratio 2:1. Conclusion and Future Directions: PBNK can be significantly induced following modified K562 stimulation. Anti-CD20 CAR enhances PBNK anti-tumor activity against CD20+ Ramos. Future directions include further characterizing the cytotoxicity activity of Anti-CD20 CAR engineered expanded PBNK cells against L/L in-vitro and survival in xenogafted mice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3511. doi:1538-7445.AM2012-3511