In order to mimic the neutralizing potency of polyclonal antibodies, combinations of neutralizing monoclonal antibodies (mAbs) are used. The main pre-requisite of any preparation which is comprised of several neutralizing mAb is that its antibodies will target distinct epitopes on the antigen molecule. The aim of this study was to elucidate a mixture of antibodies that exhibits a synergistic protective-antigen (PA)-neutralizing activity. We have previously demonstrated that mab29 has a potent PA-neutralizing activity, and that its chimeric form is able to confer full protection against anthrax infection. Here, PA-binding competition assays were applied in order to screen and isolate mAb that recognize different epitopes on PA and can bind the toxin simultaneously with mab29. Two antibodies were selected in this screen, mab33 and mab55, which have demonstrated high neutralizing activity when tested in the anthrax lethal toxin neutralization assay. In order to define the dose-effect relationships of the combination of these antibodies, the multiple-drug effect analysis was applied. It was found that combining mab29 with mab55 results in a slightly synergistic effect (combination index ≤0.9). In contrast, combining mab29 with mab33 resulted in an antagonistic effect (combination index >1.2), although when tested alone in vivo, mab33 provides full protection of rats against lethal toxin challenge. The results presented in this study generally support the notion that a cocktail of neutralizing antibodies can result in an improved activity, and suggest that the combination of mab29 with mab55 may expand the breadth of protection against anthrax infection.
