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Human Monoclonal Antibodies as a Countermeasure Against Botulinum Toxins
Final rept. 1 Mar 2011-31 Aug 2012
MORPHOTEK INC EXTON PA
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In this report, we summarize our studies to find novel, neutralizing antibodies against BoNTE, as well as the investigation into methods to generate cell lines expressing neutralizing antibodies or antibody-like molecules with specificities against BoNTA, B, and E. Through a series of rat and rabbit immunizations, we generated twelve recombinant MAbs capable of neutralizing 100LD50s of BoNTE in murine models. In parallel, multiple methods using both pro- and eukaryotic expression systems were tested for their ability to express neutralizing MAbs or MAb-like scaffolds with specificities against all three BoNTs. One system showing efficacy involved the generation of IgG-based expression constructs in which the full length light and heavy chains were expressed as a single amino acid sequence using a glycine-serine46 linker. Co-transfection of previously generated humanized BoNTA and B clones using this linker system in conjunction with dual selection markers showed that it was possible to generate a stable 293F cell line secreting both MAbs, which retained specificity to both toxins. Additionally, this material could be purified using traditional methods and mass spectrometry MS revealed both homo- and heterodimer pairing of the single chains via the hinge region of IgG. Lastly, two of the twelve BoNTE neutralizing MAbs were humanized and linked in a similar manner and tested for in vivo efficacy. One clone 199B13 was able to completely neutralize 100LD50s of BoNTE. This confirms the concept of the light-heavy chain linked IgG system and will permit the generation of a single production cell line generating material capable of neutralizing BoNTA, B, and E.
APPROVED FOR PUBLIC RELEASE