Accession Number : ADA581581


Title :   Human Monoclonal Antibodies as a Countermeasure Against Botulinum Toxins


Descriptive Note : Final rept. 1 Mar 2011-31 Aug 2012


Corporate Author : MORPHOTEK INC EXTON PA


Personal Author(s) : Kline, Brad ; Grasso, Luigi ; Kennedy, Rina ; Martinez, Bryon ; Albone, Earl ; Chao, Qimin


Full Text : https://apps.dtic.mil/dtic/tr/fulltext/u2/a581581.pdf


Report Date : 30 Nov 2012


Pagination or Media Count : 29


Abstract : 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 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-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 homo- and 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.


Descriptors :   *ANTIBODIES , *CLOSTRIDIUM BOTULINUM , CELLS(BIOLOGY) , ESCHERICHIA COLI , GENE EXPRESSION , IMMUNIZATION , IMMUNOGLOBULINS , IN VIVO ANALYSIS , MASS SPECTROMETRY , RABBITS , RATS


Subject Categories : Biochemistry
      Anatomy and Physiology
      Medicine and Medical Research
      Microbiology
      Pharmacology


Distribution Statement : APPROVED FOR PUBLIC RELEASE