Andrady, C; (2014) Pharmacokinetic and Pharmacodynamic Challenges of Antibody-Directed Enzyme Prodrug Therapy (ADEPT). Doctoral thesis , UCL (University College London).

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Abstract

Antibody-Directed Enzyme Prodrug Therapy (ADEPT) is an experimental cancer treatment. First, an antibody-enzyme is targeted to the tumour. After clearance from healthy tissue, a prodrug is administered and activated by the enzyme. MFE-CP, an anti-carcinoembryonic antibody fragment fused to the enzyme carboxypeptidase G2, has been used for ADEPT in combination with a nitrogen mustard prodrug. Clinical trials are encouraging but highlight major challenges of sub-optimal MFE-CP pharmacokinetics and drug resistance. This thesis explores means to address these challenges. MFE-CP, manufactured in P. pastoris, clears rapidly from the circulation due to yeast mannosylation. This leads to excellent tumour:blood ratios but decreased opportunity for tumour uptake. MFE-CP was therefore mutated in an attempt to reduce glycosylation. Initially, N-linked glycosylated asparagine residues were mutated to glutamine. The enzyme remained active but cleared rapidly in vivo. O-linked residue mutations were then explored. Changes that would least impede enzyme function were predicted using bioinformatics and a series of mutated constructs generated. The T55V mutation generated a functional enzyme that also cleared rapidly in vivo. The DNA damage response was investigated as a mechanism of drug resistance. Using the comet assay, DNA interstrand cross-links were shown to form rapidly in carcinoma cells and xenografts in response to ADEPT, but these were unhooked over 48 hours. The γ-H2AX and RAD51 response indicated unhooking was due to DNA damage repair. Cell cycle studies showed that ADEPT treatment also led to G2/M arrest. G2/M arrest allowed DNA repair to occur and it was hypothesised that ADEPT could be made more effective by blocking arrest and driving entry into mitosis. To test this hypothesis, Chk1 inhibitors, UCN-01 and PF-477736, were investigated. Results demonstrated an enhanced anticancer effect with the ADEPT and PF-477736 combination, whereby increased cell death was observed at 48 hours post treatment. The approach has potential for clinical translation.

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