Abstract
The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper-immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper-sensitivity reactions. New-generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti-botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400-mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab′)2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper-immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti-botulinum toxin polyclonal horse F(ab′)2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal-based to human-based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins.
Highlights
Antibodies (Abs) are the most prevalent among new pharmaceuticals approved in the last two decades [1]
To examine the role of the Fc fragment in homologous Ab neutralization, F(ab0 )2 fragments were prepared from three individual mouse anti-Botulinum neurotoxins (BoNTs)/A monoclonal antibodies (MAbs) and directly compared to their respective
F(ab0 )2 fragment were tested, similar titers were measured (IgG/F(ab0 )2 titer ratios ranging from 0.8–1 (Table 1), confirming that functional binding was not impaired by Fc fragment removal
Summary
Antibodies (Abs) are the most prevalent among new pharmaceuticals approved in the last two decades [1]. Development of “second-generation” antitoxins is underway [18,19,20] These preparations are based on combinations of either human-origin or humanized MAbs that are intended to facilitate improved pharmacokinetics along with reduced potential side effects associated with the injection of heterologous horse Abs [5,18,20]. We further elucidated the direct contribution of homologous Fc fragment to Ab-mediated toxin neutralization by systematic and quantitative evaluation of the potency and efficacy of intact and Fc-removed anti BoNT MAb and PAb preparations. The experimental design was based on the comparison of the F(ab0 ) fragment and the corresponding IgG forms of individual and combined preparations of anti-botulinum MAbs in mice and the differential neutralization potencies measured for homologous and heterologous hyper-immune PAb preparations in both in vivo and in vitro systems. Homologous Fc was shown to be essential for in vivo but not in vitro optimal neutralization by PAb preparations, and the addition of a homologous Fc arm to a heterologous pharmaceutical horse F(ab0 ) fragment preparation improved neutralization efficacy when the fragment preparation was administered to intoxicated symptomatic mice
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