Human antibodies neutralizing diphtheria toxin in vitro and in vivo

Abstract

Diphtheria is an infectious disease caused by Corynebacterium diphtheriae. the bacterium primarily infects the throat and upper airways and the produced diphtheria toxin (Dt), which binds to the elongation factor 2 and blocks protein synthesis, can spread through the bloodstream and affect organs, such as the heart and kidneys. For more than 125 years, the therapy against diphtheria has been based on polyclonal horse sera directed against Dt (diphtheria antitoxin; DAt). Animal sera have many disadvantages including serum sickness, batch-to-batch variation in quality and the use of animals for production. In this work, 400 human recombinant antibodies were generated against DT from two different phage display panning strategies using a human immune library. A panning in microtiter plates resulted in 22 unique in vitro neutralizing antibodies and a panning in solution combined with a functional neutralization screening resulted in 268 in vitro neutralizing antibodies. 61 unique antibodies were further characterized as scFv-Fc with 35 produced as fully human IgG1. The best in vitro neutralizing antibody showed an estimated relative potency of 454 IU/mg and minimal effective dose 50% (MED50%) of 3.0 pM at a constant amount of DT (4x minimal cytopathic dose) in the IgG format. The targeted domains of the 35 antibodies were analyzed by immunoblot and by epitope mapping using phage display. All three Dt domains (enzymatic domain, translocation domain and receptor binding domain) are targets for neutralizing antibodies. When toxin neutralization assays were performed at higher toxin dose levels, the neutralizing capacity of individual antibodies was markedly reduced but this was largely compensated for by using two or more antibodies in combination, resulting in a potency of 79.4 IU/mg in the in vivo intradermal challenge assay. these recombinant antibody combinations are candidates for further clinical and regulatory development to replace equine DAt.Diphtheria is an infectious disease and caused by Corynebacterium diphtheriae. The bacterium primarily infects the throat and upper airways but can also affect other body sites. The produced toxin can spread through the bloodstream and affect organs, such as the heart and kidneys. In severe cases, diphtheria toxin (DT) may cause myocarditis or peripheral neuropathy. Due to a membrane of dead tissue over the throat and tonsils, swallowing and breathing can be difficult. The disease is spread through direct physical contact or by coughing or sneezing of infected individuals 1-3 . Diphtheria is fatal in 5-10% of cases, but children under the age of five have a mortality rate of up to 20%. Treatment involves antibiotics to kill the bacteria (erythromycin or penicillin for 14 days) and administering of diphtheria antitoxin (DAT) to neutralize the effects of the toxin 4-6 . C. diphtheriae was identified as the causative agent of diphtheria in 1883 and in 1888 the diphtheria toxin was first described in the culture medium of C. diphtheriae 7 . The gene for DT is encoded on a coryn...

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