The gene for the human recombinant
eosinophil-derived neurotoxin (rEDN) was synthesized and fused to the gene encoding a single chain antibody (sFv) to the human
transferrin receptor (
EDNsFv). Both rEDN and
EDNsFv were expressed as insoluble
proteins in inclusion bodies in Escherichia coli BL21(DE3). Following denaturation and renaturation, EDN and
EDNsFv were partially purified by chromatography on
heparin-Sepharose. Final purification of EDN was achieved by
Sephadex G-100, whereas
EDNsFv which contained a 6-histidyl residue carboxyl terminus was highly purified using the
metal chelate resin, Ni(2+)-nitriloacetic
acid. Whereas the recombinant EDN had
ribonuclease activity that was similar to the native
protein, the fusion
protein had enzymatic activity that was 6-13% that of native EDN. The fusion
protein was able to bind to the human
transferrin receptor. In contrast to rEDN that had no inherent cytotoxicity to human
tumor cells, the
EDNsFv fusion
protein was cytotoxic to human
leukemia cells that express the human
transferrin receptor with an IC50, 0.2-1 nM. At 1.3 nM
EDNsFv, no cytotoxicity was observed on cells that lack the human
transferrin receptor. Free antibody to the human
transferrin receptor, E6, inhibited the cytotoxicity of the
EDNsFv. Human
enzymes may be engineered to acquire cytotoxic properties by fusing them to
antibodies. Thus, they may be candidates for the construction of immunofusion
proteins that may be less immunogenic than
immunotoxins containing bacterial- or plant-derived toxin moieties.