Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells
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Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcγRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3ζ chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.
KeywordsCD20 scFv antibody Natural killer cell Chimeric antigen receptor Adoptive therapy
We thank Dr. Barbara Schnierle for providing pEFIRES-P vector, Dr. Annette Romanski for BV173 and NALM-6 cells, Dr. Byoung S. Kwon for anti-4-1BB antibody BBK-1, Daniela Bott for isolation of primary B and NK cells, Dr. Brigitte Rüster for help with microscopical analysis, Dipl. Ing. Nicola Krzossok for help with NK-92 cytotoxicity assays, Dr. Boris Brill, Sabrina Lehmen and Christiane Peter for help with animal experiments, and Dr. Markus Biburger for helpful suggestions. This work was supported in part by research grant 102386/10-2244 from Deutsche Krebshilfe. G. Maki was supported by grant CLL-63119, Section of Hematology, Rush University Medical Center.
Supplementary movie Selectivity and kinetics of target cell killing. NIH3T3-CD20 cells transduced with a retroviral vector encoding enhanced green fluorescent protein (eGFP) were mixed at a 1:1 ratio with parental NIH3T3 cells and grown overnight. Then NK-92-scFv(Leu-16)-ζ cells were added at an effector to target ratio of 1:1, microscopic images of a single field were taken at 1.5 min intervals for 6.4 h, and assembled into a QuickTime movie at 10 frames per second. At the beginning of the movie, a fluorescence microscopic image of eGFP- and CD20-positive NIH3T3-CD20(eGFP), and eGFP- and CD20-negative NIH3T3 cells before addition of NK cells is shown. Exemplary NIH3T3-CD20(eGFP) cells are indicated by white circles, exemplary parental NIH3T3 cells by black arrows. Selected images from this experiment are also shown in Fig. 5. (MOV 2.33 mb)
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