The continuously growing Natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origin without affecting normal human cells. It is the only NK cell line that has entered clinical trials to date. Acute lymphoblastic leukemias (ALL) show variable sensitivity towards NK cells including NK-92-lysis. All T-ALL cell lines tested (MOLT-3, MOLT-4 and CEM-T) display moderate sensitivity to NK cytotoxicity with approximately 22±2 – 24±2% killing at an effector to target (E:T) ratio of 10:1, respectively. Comparable results were obtained with primary patient derived T-ALLs (n=4), displaying specific killing rates of 17±5 – 20±2%. In contrast, B lineage ALL cell lines (NALM-6, SEM, REH, JKB, BV173, SupB15, TOM-1, TMD5) and cells derived from patients (n=11) were resistant to NK-92-mediated lysis, with specific killing rates ranging from 2±1% to 13±3% at an E:T ratio of 10:1. Approaches to overcome resistance of B-precursor ALL by re-targeting may shed light on the specific reasonable mechanism conferring resistance and lead to novel therapeutic strategies. Here we have generated genetically modified NK-92 cells expressing a chimeric antigen receptor specific for the pan B cell antigen CD19 which is universally expressed by B lineage leukemia cells. This receptor fragment kindly provided by H. Zola (Child Health Research Institute North Adelaide, Australia) consists of the CD19 specific scFv(CD19) antibody fragment, a flexible hinge region, the CD3 ζ chain and a Myc-tag. Transduced cells were selected with G418, and surface expression of the chimeric scFv(CD19)-ζ construct was verified by FACS analysis using the Myc-tag-specific mAb 9E10. No difference in cytotoxic activity of NK-92 and transduced NK-92-scFv(CD19)-ζ cells towards CD19 negative targets (K562, MOLT-4) was found. In contrast, NK-92-scFv(CD19)-ζ cells specifically and efficiently lysed CD19 expressing B precursor leukemia cells (NALM-6, SEM, B173, SupB15, TOM-1, TMD5) including cells that were completely resistant to cytolytic activity of parental NK-92 cells. Killing of SupB15 and BV173 cells was 10,0±0,8% and 12,0±8,5% with parental NK92 vs. 58,2±14,8% and 52,3±4,1% lysis with NK92 cells expressing the CD19 specific scFv(CD19) antibody fragment. These results demonstrate that efficient retargeting of NK-cell cytotoxicity can be achieved, and might allow the generation of potent cell-based therapeutics. Since the widespread resistance of B precursor ALL blasts to NK cell cytotoxicity is not caused by mechanisms commonly relevant in tumors, this model may allow dissection of possible mechanisms of resistance.

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