Protection of Mice Against Tetanus Toxin by Combination of Two Human Monoclonal Antibodies Recognizing Distinct Epitopes on the Toxin Molecule

Abstract

Human B-lymphocytes were fused with the human lymphoblastoid B-cell line WI-L2-729 HF2. Hybridoma frequencies were in the range of 10^-5 when the mononuclear cells were (a) prestimulated with pokeweed mitogen (PWM), (b) fused with polyethyleneglycol (PEG), and (c) selected in a hypoxanthine-azaserin (HAza) containing medium. To generate monoclonal antibodies (MAb) specific for tetanus toxin (TToxin) human spleen cells were precultured with PWM plus tetanus toxoid (TToxoid) in two separate fusions. Two hybridomas were selected based on high binding activity using an enzyme-linked immunosorbent assay (ELISA) for TToxoid. Both hybridomas, cloned twice and designated anti-TT1 and anti-TT2, exhibited a near tetraploid karyotype and showed stable production of antibody (0.15 μg/ml) over several months. Using ELISA for fragments of TToxin and the immunoblotting technique, the two IgG₁ monoclonal antibodies were found to bind to the heavy chain portion of the B-fragment (anti-TT1) and on the C-fragment (anti-TT2) of the toxin. When tested in an ELISA with TToxin the combination of anti-TT1 and anti-TT2 showed higher binding activity than either reagent alone. In an in vivo neutralization assay mice were completely protected against TToxin by the combination of the two antibodies while either antibody alone resulted only in a delay of death of the mice. These findings demonstrate that a cocktail of appropriate human monoclonal antibodies can be far superior to a single reagent when used in a therapeutic setting.