An oligoclonal combination of human monoclonal antibodies able to neutralize tetanus toxin in vivo

Abstract

The use of antibody-based therapy to treat a variety of diseases and conditions is well documented. The use of antibodies as an antidote to treat tetanus infections was one of the first examples of immunotherapy and remains the standard of care for cases involving potential infections. Plasma-derived immunoglobulins obtained from human or horse pose risks of infection from undetectable emergent viruses or may cause anaphylaxis. Further, there is a lack of consistency between lots. In the search for new formulations, we obtained a series of clonally related human monoclonal antibodies (mAbs) derived from B cells sorted from donors that presented anti-tetanus neutralizing titers. Donors were revaccinated prior to blood collection. Different strategies were used for single-cell sorting, since it was challenging to identify cells at a very low frequency: memory B cell sorting using fluorescent-labeled tetanus toxoid and toxin as baits, and plasmablast sorting done shortly after revaccination. Screening of the recombinant mAbs with the whole tetanus toxin allowed us to select candidates with therapeutic potential, since mAbs to different domains can contribute additively to the neutralizing effect. Because of selective binding to different domains, we tested mAbs individually, or in mixtures of two or three, in the neutralizing in vivo assay specified by Pharmacopeia for the determination of polyclonal hyperimmune sera potency. An oligoclonal mixture of three human mAbs completely neutralized the toxin injected in the animals, signaling an important step for clinical mAb development.

Keywords: Epitope mapping; Ganglioside; Neuron receptor; Neutralizing antibody; Peptide array; Tetanus toxin.

Fig. 1

Flow cytometry for detection of TeNT-specific B cells (A) or plasmablasts (B). (1) Gating strategy for detection of lymphocytes based on forward scatter (FSC) and side scatter (SSC); (2) Doublets were excluded using FSC height versus FSC area; (A3) Ig class-switched B cells were defined as CD19⁺IgG⁺; (A4) TeNT-specific B cells were defined as double-positive TeNT-Biotin/Streptavidin-PerCP-Cy5.5⁺/TeNT-Alexa Fluor 647⁺; (B3) Live cells were defined as DAPI⁻; (B4) B cells were defined as CD19⁺; (B5) Plasmablasts were defined as CD27⁺CD38⁺.

Fig. 2

Graphs showing the repertoire of the VH fragments (A) and the number of somatic mutations (B) in the clonally related sequences. Cells were isolated by two different strategies to stain memory cells (CD19⁺IgG⁺TeNT⁺) and plasmablasts (CD19⁺CD27⁺CD38⁺).

Fig. 3

Monoclonal antibodies reactivity by ELISA using 96-wells plates coated with (A) 5 μg/mL tetanus toxin, (B) 5 μg/mL tetanus toxoid, and (C) 2 μg/mL recombinant tetanus toxin fragment C. (D) mAbs capacity to inhibit TeNT binding was assessed by incubating GT1b with an appropriate level of TeNT alone or with increasing concentrations of each mAb. The mAbs were tested in the range from 0.4 to 50 ng/mL. Serum of a vaccinated person was used as positive control for A-C; horse hyperimmune serum was used in assay D.

Fig. 4

Western Blotting showing the recognition of different TeNT chains by the mAbs. In (A), SDS-PAGE of the TeNT under nonreducing (lane 1) and reducing (lane 3) conditions, showing the entire TeNT (a), the heavy (b), and light (c) chains. BSA (lane 2) was used as negative control. M: protein ladder. In (B), results of WB: BUT-TT-140-08 e BUT-TT-117-08 recognized the heavy chain (b); BUT-TT-120-10 recognized the light chain (c) and the entire TeNT (a), which was expected since it also contains the light fragment; BUT-TT-243-10 recognized the heavy chain (b) and the entire TeNT (a); and BUT-TT-143-10 recognized only the non-processed (entire) TeNT.

Fig. 5

In vivo tetanus toxin neutralizing assay. The in vivo neutralizing capacity of TeNT-specific mAbs was evaluated using Swiss mice in accordance to the potency test used for approval of hyperimmune horse sera. A fixed amount of reference TeNT was incubated with the serially diluted mAbs (alone or mixed) and injected subcutaneously into 10 mice for each dilution. The values indicated in the legend correspond to the amount of mAb injected in each one of 10 animals. After four days of observation for any signs of tetanus the survival rates were calculated according to a probit statistical model.

Fig. 6

Representations of Tetanus Toxin (TeNT) structure and mAbs epitopes detected by epitope array. Light chain (active domain) is marked in blue, heavy chain is marked in green for Translocation domain and in pink for Binding domain (Fragment C). The epitopes recognized by each mAb in the peptide array are shown in yellow. Full view: Surface representations showing epitope accessibility. Close view: epitopes are shown in stick representations. The representations were generated based on the Crystal structure of TeNT (PDB: 5n0b). The figure was produced using PyMol software.