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A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I
H Linardou1, A A Epenetos, M P Deonarain
1Oncology Unit, Department of Clinical Oncology, Imperial Cancer Research Fund, Imperial College Medical School at Hammersmith Campus, London, UK.
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.
Insights
Researchers developed a novel chimeric molecule combining a cancer-targeting antibody fragment with Deoxyribonuclease-I (DNase-I) for potential tumor therapy. This engineered DNase-I demonstrated potent, targeted cancer cell killing in vitro, offering a promising new strategy for cancer treatment.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapy
Background:
- Mammalian proteins are explored for targeted tumor therapy.
- Deoxyribonuclease-I (DNase-I) is a DNA-degrading enzyme with potential for tumor targeting due to its selective cytotoxicity when nuclear-localized.
Purpose of the Study:
- To investigate the cytotoxic potential of mammalian DNase-I.
- To explore its use in tumor-targeting strategies for cancer therapy.
Main Methods:
- Designed and constructed a chimeric molecule: anti-PLAP scFv fused to bovine pancreatic DNase-I.
- Developed a bacterial expression system for DNase-I and the chimera.
- Produced and purified active DNase-I and the anti-PLAP scFv-DNase-I chimera.
- Characterized the chimera's antigen-binding and DNA-degrading activities in vitro.
Main Results:
- Successfully produced and purified active DNase-I and the chimeric molecule.
- The anti-PLAP scFv-DNase-I chimera exhibited both antigen-binding and DNA-degrading activities.
- The chimeric molecule demonstrated high in vitro cytotoxicity against cells expressing the PLAP antigen.
Conclusions:
- Targeting mammalian DNase-I offers a novel strategy for selective cancer cell killing.
- This approach promises reduced systemic toxicity and immunogenicity compared to current immunotoxins.
- The engineered DNase-I chimera shows potential for effective and safer cancer therapy.