Human monoclonal antibodies as a countermeasure against Botulinum toxins

In this report, we summarize our studies to find novel, neutralizing antibodies against BoNT/E, as well as the investigation into methods to generate cell lines expressing neutralizing antibodies or antibody-like molecules with specificities against BoNT/A, /B, and /E. Through a series of rat and rabbit immunizations, we generated twelve recombinant MAbs capable of neutralizing 100LD50s of BoNT/E in murine models. In parallel, multiple methods using both proand 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-serine4)6 linker. Co-transfection of previously generated humanized BoNT/A 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 homoand heterodimer pairing of the single chains via the hinge region of IgG. Lastly, two of the twelve BoNT/E 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 BoNT/E. 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 BoNT/A, /B, and /E. (a) Papers published in peer-reviewed journals (N/A for none) Enter List of papers submitted or published that acknowledge ARO support from the start of the project to the date of this printing. List the papers, including journal references, in the following categories: