A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I.

ABSTRACT

A number of mammalian proteins with suitable biological activities have been considered for use in targeted tumour therapy. Deoxyribonuclease-I (DNase-I), an endonuclease that degrades double-stranded DNA, represents an attractive candidate for tumour targeting since it is normally non-toxic yet could be highly cytotoxic when redirected to the cell nucleus. Our aim was to investigate the cytotoxic potential of mammalian DNase-I and its possible use in tumour-targeting strategies for cancer therapy. A chimeric molecule comprising a scFv reactive against the human placental alkaline phosphatase (hPLAP) and bovine pancreatic DNase-I was designed and investigated. The development of a tightly controlled system for the bacterial expression of DNase-I and its chimera is described. The production and purification of active DNase-I from the soluble cell fraction and significant yields from the insoluble fraction by isolation and refolding are described. The construction, expression, purification and in vitro characterisation of an anti-PLAP scFv-DNase-I chimera is also described. This molecule was shown to possess both antigen-binding and DNA-degrading activity in in vitro assays, thus combining the specific cell-targeting properties of the scFv and the potent, highly catalytic activity of the endonuclease. Furthermore, this chimeric molecule was highly cytotoxic in vitro in cells expressing the PLAP antigen. Targeting mammalian DNase-I provides a novel therapeutic strategy for selective cell killing, with the promise of less systemic toxicity and immunogenicity than currently used immunotoxins.