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As a positive control we used MV-H82-EGFR

As a positive control we used MV-H82-EGFR.scFv, whose H protein Talnetant is modified with a C-terminal EGFR scFv/His x6 domain allowing it to be retargeted to EGFR and HIS scFv expressed on Vero-His cells. [21, 22] and over expressed on several types of cancer [23C25] and CD46 which is a cellular receptor for laboratory-adapted MV strains [26]. CD46 is a regulator of complement activation [26, 27] that is ubiquitously expressed on all human nucleated cells and over expressed on many different cancer cell types making them highly susceptible to MV-Edm infection and its cytopathic effects [28]. MV-Edm can be retargeted to specific tumor cells by linking a single-chain antibody (single chain Talnetant fragment variable, scFv) or naturally occurring ligand to the virus attachment hemagglutinin (H) glycoprotein displayed on the virus surface. The ablation of receptor CD46 and SLAM binding sites limits virus attachment and entry to cells expressing the receptor for the scFv or ligand linked to H. Retargeted MV-Edm derivatives retain their oncolytic activity against xenografts expressing target receptors [29C37]. A variety of scFvs have been displayed on H against different receptors: EGFR (epidermal growth factor receptor) [29, 31]; EGFRvIII [29, 32]; HER2/neu (HER2: Human Epidermal Growth Factor Talnetant Receptor 2) [38], CD20 [36, 37]; folate receptor alpha [33]; CD38 [29]; CEA (carcinoembryonic antigen) [39], prostate-specific membrane antigen (PSMA) [40] and an unidentified receptor over-expressed on multiple myeloma cells that can be targeted by Wue scFv [35]. Ligands linked to H have also successfully redirected entry, for example: amino-terminal fragment of urokinase plasminogen activator (uPA) targeting uPA receptor on breast tumors and tumor stroma [34]; snake venom peptide echistatin, targeting integrins v3 and 51 expressed on vascular endothelium [41]; single-chain T-cell receptor (scTCR) targeting a specific peptide/MHC complex [42] and interleukin-13 targeting gliomas [30]. One of the major hurdles for oncolytic virotherapy is pre-existing immunity against the oncolytic virus [43, 44]. Measles oncolytic virotherapy is limited by preexisting immunity due to widespread global vaccination against measles [45]. The hemaggluntinin attachment protein is the major target for neutralizing antibodies [46] that tend to cluster at the receptor Talnetant binding surface targeting a conserved neutralizing antigenic region ABL [47C51]. Retargeted MV derivatives have two modifications that could potentially destroy or shield epitopes within the receptor-binding surface. The first modification is a set of two (Y481A and R533A) or four (Y481A, R533A, S548L and F549S) mutations that ablate infection via CD46 and SLAM [29]. The second modification is the scFv or ligand linked to the H C-terminus used to retarget MV to specific receptors. This additional polypeptide domain could shield one or more antibody epitopes and protect the virus from neutralization [52]. Should the utility of retargeted oncolytic MVs extend to evasion of serum neutralization it would render them superior to MV derivatives currently tested clinically. In this study we used chimeric H proteins with and without mutations that ablate MV receptor binding to determine if these mutations protect MV-Edm from mAbs targeting the mutated receptor-binding surface. We investigated if the displayed domain can shield mAb epitope(s) and if the size of the domain determines how well an epitope is protected. We then addressed the question if retargeted MV derivatives evade human serum neutralization, since entry is no longer dependent on H binding MV receptors, but is mediated by a separate polypeptide domain attached to the H C-terminus by a linker. Our data demonstrate that mutations that ablate CD46 and SLAM binding protect retargeted MV from mAbs targeting the receptor binding-surface but not from human serum neutralization. The displayed domain provided no significant additional protection from neutralizing antibodies tested. MATERIALS AND METHODS Cell Culture Retargeted MVs were propagated and titered on Vero Cells (African green monkey kidney cells) stably expressing membrane-anchored single-chain antibody that recognizes a six-histidine peptide (Vero-His), described previously [29, 53]. Vero-His cells Talnetant were grown in Dulbeccos Modified Eagles Medium (DMEM) with 5% Fetal Bovine Serum (FBS)..