Discussion

Receptor-mediated nutrient uptake in trypanosomes involves internalisation of surface receptors and their ligands through the endosomal pathway. Toxin-conjugated monoclonal antibodies recognising epitopes on such receptors should therefore be internalised by the trypanosome, providing targeted delivery of toxins into the parasite. To test this hypothesis, the T. b. brucei haptoglobin-haemoglobin receptor (HpHbR) was selected as a target receptor. The HpHbR is a low copy number GPI-anchored surface protein that functions in haem acquisition through internalisation of host HpHb and in innate immunity through internalisation of trypanosome lytic factor 1. Single chain variable fragments that recognise recombinant HpHbR N-terminal domain were selected by phage-display then converted into humanised IgGs and fluorescently labelled. Five out of seven selected antibodies were efficiently and specifically internalised into T. b. brucei wild-type cells and not into HpHbR null cells. Conjugation of these monoclonal HpHbR IgGs to pyrrolobenzodiazepine (PBD) toxins led to targeted cell killing of trypanosomes, including human infective T. b. rhodesiense, at picomolar concentrations. This work demonstrates the potential for exploitation of receptor-mediated nutrient uptake for targeted delivery of toxins into African trypanosomes using antibody-toxin conjugates.