The lipopolysaccharide (LPS) of Francisella tularensis (Ft), the Gram negative bacterium that causes tularemia, has been shown to be a main protective antigen in mice and humans; we have previously demonstrated that murine anti-Ft LPS IgG2a monoclonal antibodies (MAbs) can protect mice against otherwise lethal intranasal infection with the Ft live vaccine strain (LVS). Here we show that four IgG2a anti-LPS MAbs are specific for the O-polysaccharide (O-antigen [OAg]) of Ft LPS. But whereas three of the MAbs bind to immunodominant repeating internal epitopes, one binds to a unique terminal epitope of Ft OAg. This was deduced from its even binding to both long and short chains of the LPS ladder in Western blots, its rapid decrease in ELISA binding to decreasing solid-phase LPS concentrations, its inability to compete for LPS binding with a representative of the other three MAbs, and its inability to immunoprecipitate OAg despite its superior agglutination titer. Biacore analysis showed the end-binding MAb to have higher bivalent avidity for Ft OAg than the internal-binding MAbs and provided an immunogenicity explanation for the predominance of internal-binding anti-Ft OAg MAbs. These findings demonstrate that non-overlapping epitopes can be targeted by antibodies to Ft OAg, which may inform the design of vaccines and immunotherapies against tularemia.
We describe the expression and consistent production of a first target-specific recombinant human polyclonal antibody. An anti-Rhesus D recombinant polyclonal antibody, Sym001, comprised of 25 unique human IgG1 antibodies, was produced by the novel Sympress expression technology. This strategy is based on site-specific integration of antibody genes in CHO cells, using the FRT/Flp-In recombinase system. This allows integration of the expression construct at the same genomic site in the host cells, thereby reducing genomic position effects. Different bioreactor batches of Sym001 displayed highly consistent manufacturing yield, antibody composition, binding potency, and functional activity. The results demonstrate that diverse recombinant human polyclonal antibody compositions can be reproducibly generated under conditions directly applicable to industrial manufacturing settings and present a first recombinant polyclonal antibody which could be used for treatment of hemolytic disease of the newborn and/or idiopathic thrombocytopenic purpura.
Tularemia is caused by the Gram-negative facultative intracellular bacterium Francisella tularensis, which has been classified as a Category A Select Agent -a likely bioweapon. The high virulence of F. tularensis and the threat of engineered antibiotic resistant variants warrant the development of new therapies to combat this disease. We have characterized 14 anti-Francisella hybridoma antibodies derived from mice infected with F. tularensis live vaccine strain (LVS) for potential use as immunotherapy of tularemia. All 14 antibodies cross-reacted with virulent F. tularensis type A clinical isolates, eight bound to a purified preparation of LVS LPS, and six bound to five protein antigens, identified by proteome microarray analysis. An IgG2a antibody, reactive with the LPS preparation, conferred full protection when administered either systemically or intranasally to BALB/c mice post challenge with a lethal dose of intranasal LVS; three other antibodies prolonged survival. These anti-Francisella hybridoma antibodies could be converted to chimeric versions with mouse V regions and human C regions to serve as components of a recombinant polyclonal antibody for clinical testing as immunotherapy of tularemia. The current study is the first to employ proteome microarrays to identify the target antigens of anti-Francisella monoclonal antibodies and the first to demonstrate the systemic and intranasal efficacy of monoclonal antibodies for post exposure treatment of respiratory tularemia.
Communicated by Alfred Nisonoff, April 9, 1990 (received for review February 22, 1990
ABSTRACTThe basis for the 200-fold difference in affinity between two hybridoma antibodies specific for the hapten p-azophenylarsonate (Ars) that have diversified by somatic hypermutation was examined. Oligonucleotide-directed mutagenesis was used to sequentially convert the nucleotide sequence ofthe lower-affinity antibody into that ofthe higher-affinity one, and the mutant antibodies generated by transfection of hybridoma cells were analyzed for affinity to Ars-tyrosine. The data
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