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Directed Enzyme Prodrug Therapy (ADEPT) University College London Pharmacokinetic and Pharmacodynamic Challenges of Antibody- Directed Enzyme Prodrug Therapy (ADEPT) A thesis submitted to the University College London (UCL) for the degree of Doctor of Philosophy in the faculty of Biomedical Sciences, Department of Oncology, UCL Cancer Institute, UCL Carima Andrady 2014 Declaration of Originality ‘I Carima Andrady confirm that the work presented in this thesis is my own. Where information has been derived from other sources; I confirm that this has been indicated in the thesis.’ 1 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. 2 Contents Abstract .................................................................................................. 2 Contents ................................................................................................. 3 List of Figures ........................................................................................ 8 List of Tables ....................................................................................... 11 List of Abbreviations ............................................................................ 12 Acknowledgements .............................................................................. 15 1 Introduction .................................................................................... 17 1.1 Cancer ................................................................................................. 17 1.2 Antibodies for cancer therapy ............................................................ 18 1.2.1 Antibody structure ................................................................................ 18 1.2.2 Engineering antibodies for therapy ...................................................... 20 1.3 Recombinant antibodies ..................................................................... 25 1.3.1 Recombinant technology ...................................................................... 25 1.3.1.1 Phage display ................................................................................ 25 1.3.1.2 Transgenic mice ............................................................................ 26 1.3.2 Recombinant antibody fragments ........................................................ 27 1.3.2.1 ScFv .............................................................................................. 27 1.4 Modified antibodies for targeted therapy ........................................... 30 1.4.1 Radioimmunotherapy (RIT) ................................................................. 30 1.4.2 Immunotoxins ...................................................................................... 30 1.4.3 Antibody-drug conjugates .................................................................... 31 1.4.4 Antibody-directed enzyme prodrug therapy ........................................ 31 1.5 ADEPT ............................................................................................... 32 1.5.1 Carboxypeptidase G2 ADEPT system ................................................. 38 1.5.1.1 Chemical conjugate....................................................................... 38 1.5.1.2 Chemical conjugate clinical trials ................................................. 39 1.5.1.3 Recombinant fusion protein .......................................................... 40 1.5.1.4 Fusion protein clinical trial ........................................................... 41 1.5.2 Current challenges of ADEPT ............................................................. 42 1.6 ADEPT Pharmacokinetics: Clearance mechanisms .......................... 43 3 1.6.1 Second antibody systems ..................................................................... 44 1.6.2 Glycosylation ....................................................................................... 46 1.6.2.1 Protein glycosylation by yeast P. pastoris .................................... 47 1.7 ADEPT Pharmacodynamics: DNA damage response ....................... 49 1.7.1 ADEPT-induced DNA damage ............................................................ 50 1.7.2 The DNA damage response (DDR) ..................................................... 51 1.7.3 Repair of DNA ICLs ............................................................................ 52 1.7.4 Repair of ICL-associated DSBs ........................................................... 54 1.7.4.1 Gamma-H2AX .............................................................................. 55 1.7.4.2 RAD51 .......................................................................................... 56 1.8 Thesis Aims ........................................................................................ 57 2 Materials and Methods ................................................................... 59 2.1 Materials ............................................................................................. 59 2.1.1 Molecular Biology Reagents ................................................................ 59 2.1.1.1 Primers and enzymes .................................................................... 59 2.1.1.2 Plasmids ........................................................................................ 60 2.1.1.3 Electrophoresis consumables ........................................................ 62 2.1.1.4 Microbial strains ........................................................................... 66 2.1.1.5 RT-PCR ........................................................................................ 67 2.1.2 Detection of proteins ............................................................................ 68 2.1.2.1 Antibodies ..................................................................................... 68 2.1.2.2 Buffers .......................................................................................... 70 2.1.3 Protein deglycosylation ........................................................................ 70 2.1.3.1 Deglycosylating enzymes ............................................................. 70 2.1.3.2 Chromatography buffers ............................................................... 71 2.1.4 Modified single-cell gel electrophoresis (comet) assay ....................... 71 2.1.5 Cell culture ........................................................................................... 71 2.1.5.1 ADEPT reagents ........................................................................... 72 2.1.5.2 Cell cycle inhibitors ...................................................................... 72 2.1.6 In vivo studies ....................................................................................... 73 2.2 Methods .............................................................................................. 73 2.2.1 Cloning and propagation of DNA ........................................................ 73 2.2.1.1 Plasmids ........................................................................................ 73 2.2.1.2 Plasmid digestion .......................................................................... 74 2.2.1.3 Ligation and clean-up ................................................................... 75 4 2.2.1.4 Transformation into competent bacterial cells.............................. 75 2.2.1.5 Checking for positive clones......................................................... 76 2.2.1.6 Isolation of plasmid DNA ............................................................. 76 2.2.2 Expression and purification of
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