Design, construction and evaluation of a humanised ADEPT system.
Doctoral thesis, UCL (University College London).
Antibody directed enzyme prodrug therapy (ADEPT) is a two phase system where an antibody-enzyme is targeted to tumour cells. A relatively non-toxic prodrug is administered and converted to active drug at the tumour. The system is most successful when foreign enzymes are used as this eliminates the danger of endogenous prodrug activation and toxicity to healthy tissue. However, foreign enzymes are immunogenic and this has limited the potential of ADEPT clinical trials. The central hypothesis is that a human enzyme can be mutated to reduce affinity for natural substrate and selectively activate a designed prodrug to provide a less immunogenic ADEPT treatment. To optimise pharmacokinetics, it was proposed that a clearing system was required. Human pancreatic ribonuclease was mutated to reduce the affinity for its substrate and a prodrug was designed for selective recognition by mutated RNase in preference to the wild type enzyme. The proteins were generated, expressed within inclusion bodies of E.coli and additional mutants were tested for efficacy. Prodrug and active drug toxicity was determined and turnover of prodrug established. Various dimers of a genetic fusion of mutated enzyme and a humanised single chain antibody fragment (scFv) to carcinoembryonic antigen (CEA) were created and tested in vitro. Finally, a scFv was isolated from a phage display library for generation into a clearing antibody. Results showed the active drug was >100 fold more toxic than prodrug in vitro and the prodrug was stable. The mutated RNase was 6 fold more specific for prodrug than wild type enzyme and additional mutations did not increase the affinity for prodrug. Mutant enzymes were found to be inhibited by ribonuclease inhibitor and this was addressed by generation of dimeric constructs based on bovine seminal RNase. These results support the proposed hypothesis and indicate a feasible approach to a humanised ADEPT system.
|Title:||Design, construction and evaluation of a humanised ADEPT system|
|Additional information:||Permission for digitisation not received for commercial reasons|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Wolfson Institute and Cancer Institute Administration > Cancer Institute > Research Department of Oncology|
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