A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I

A number of mammalian proteins with suitable biological activities have bee n considered for use in targeted tumour therapy. Deoxyribonuclease-I (DNase -I), an endonuclease that degrades double-stranded DNA, represents an attra ctive candidate for tumour targeting since it is normally non-toxic yet cou ld be highly cytotoxic when redirected to the cell nucleus. Our aim was to investigate the cytotoxic potential of mammalian DNase-I and its possible u se in tumour-targeting strategies for cancer therapy. A chimeric molecule c omprising a scFv reactive against the human placental alkaline phosphatase (hPLAP) and bovine pancreatic DNase-I was designed and investigated. The de velopment of a tightly controlled system for the bacterial expression of DN ase-I and its chimera is described. The production and purification of acti ve DNase-I from the soluble cell fraction and significant yields from the i nsoluble fraction by isolation and refolding are described. The constructio n, expression, purification and in vitro characterisation of an anti-FLAP s cFv-DNase-I chimera is also described. This molecule was shown to possess b oth antigen-binding and DNA-degrading activity in in vitro assays, thus com bining the specific cell-targeting properties of the scFv and the potent, h ighly catalytic activity of the endonuclease. Furthermore, this chimeric mo lecule was highly cytotoxic in vitro in cells expressing the FLAP antigen. Targeting mammalian DNase-I provides a novel therapeutic strategy for selec tive cell killing, with the promise of less systemic toxicity and immunogen icity than currently used immunotoxins. Int. J. Cancer 86:561-569, 2000. (C ) 2000 Wiley-Liss, Inc.