663. Can Oncolytic Measles Virus Targeted to CD20 Recapitulate Any of the Effects of Rituximab in the Treatment of Acute Lymphoblastic Leukemia?
Authors: Katharine Bailey, Aditi Dey, Sarah Aref, Lucile Broyer, Adele K. Fielding
Journal: Molecular Therapy (2016)

The Edmonston-B-derived, vaccine strain of measles virus (MVNSe) has oncolytic activity in numerous preclinical tumor models; safety and some efficacy has been demonstrated in phase 1 clinical trials for myeloma and ovarian carcinoma. Our work focuses on developing MV for the treatment of adult acute lymphoblastic leukaemia (ALL) - we have shown MV to be highly effective in preclinical models. An overall goal of our work is to determine how best to combine MV with other relevant, effective agents in ALL therapy. The antiCD20 monoclonal antibody rituximab improves outcome in adult ALL. Overall, we aim to determine whether targeting the CD20 antigen with a modified MV - wherein a single chain Fv antibody against CD20 is expressed as a C-terminal extension of the H glycoprotein [MVHαCD20, Bucheit et al, 2003] can recapitulate the effects of exogenously administered rituximab. A particular question in this study is whether neutrophils - which we have shown are important in mediating some of the MV-oncolytic effects and are definitely important as effector cells in rituximab antibody-dependent cellular cytotoxicity - can be as efficiently recruited by MVHαCD20 as by rituximab. We retrovirally transduced CD20 negative B-precursor ALL cell line NALM-6 cells to express human CD20. Cells were flow sorted to give 'high', and 'low' expressing populations, based on mean fluorescence intensity. Raji, a Burkitt lymphoma cell line with very high CD20 expression, was used as a positive control. We compared the oncolytic activity of MVNSe and MVHαCD20 by quantifying syncytia formation and cell death. Characteristic syncytia appeared earlier and were larger in CD20+ NALM6 cells when infected by MVHαCD20 as compared to MVNSe. However there was no significant difference between the conditions when cell death was measured after 5 days. Next, we carried out FACS-based neutrophil phagocytosis assays in which target cells were labelled with PKH67. Percentage phagocytosis was determined by the frequency of dual expressing CD15+/PKH67+ cells, relative to total CD15+ cells. We established that the'high'CD20NALM6 cells generated a significantly greater neutrophil-mediated phagocytic response to rituximab alone than the 'low'CD20NALM6 cells with a maximum of 57% at E:T ratio of 1:5. Next, we investigated neutrophil-mediated phagocytosis following MV infection. Complement was not added to the medium. At 24 hours post infection with MVNSe, there was only a background level of phagocytosis (<5%) suggesting that neutrophil-mediated phagocytosis is not stimulated by direct microbial recognition in response to MV infection. MVHαCD20 showed similar results, suggesting that the scFVantiCD20 was also unable to stimulate neutrophil-mediated phagocytosis, possibly due to lack of Fc receptor. Studies in the presence of complement are ongoing. Despite the lack of neutrophil-mediated phagocytosis, the overall cell death, as assessed by DAPI staining, was significantly greater after MVHαCD20 infection than after rituximab administration. Future studies into the differential utility of MVHαCD20 over MVNSe in ALL therapy will include work with monocytes and NK cells and co-administration of corticosteroids.