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Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Somatic mutations can lead to cancer, often by altering the activity of kinases within signaling pathways that control cell growth and proliferation. Targeted cancer therapeutics are designed and used to regulate these aberrant signaling pathways in cases where somatic mutations within kinase genes predict a positive patient response to those treatments. For example, the V600E mutation in BRAF, the gene coding for the BRAF serine threonine kinase, predicts the effectiveness of vemurafenib in treating metastatic melanoma, while the mutational status of codons G12/G13 in the KRAS gene predicts likely colorectal cancer patient response to the monoclonal antibody (mAb) cetuximab.¹-³ However, FDA approved assays currently used to detect missense mutations in BRAF V600 and KRAS G12/G13 are not capable of detecting clinically actionable mutations at mutational frequencies low enough to permit their robust application to early disease detection or minimal residual disease monitoring. Moreover, detection of all clinically actionable missense mutations is not certain or generally achieved, in part due to limitations to assay specificities and the inability to unequivocally discriminate missense mutations from synonymous germline sequence variations. This thesis addresses that limitation through the development and validation of a novel platform for creating highly sensitive assays against all possible missense mutations in an oncogenic hotspot codon or adjacent set of hotspot codons that ameliorates the known limitations to current FDA-approved assays. The platform is designed to enable development of assays against all possible missense mutations in oncogenic hotspots and, if required, unequivocally differentiate them from synonymous germline alleles. It utilizes droplet digital PCR (ddPCR) technology and chimeric wild-type specific LNA/DNA probes to create a novel “WT-negative” screening paradigm. The platform is applied to the creation of two new assays of potential clinical use in cancer diagnostics and theranostics. The first provides a reliable and sensitive screening and detection of all known clinically actionable mutations in BRAF V600, and the second achieves the same for KRAS G12/G13. Both assays show complete diagnostic accuracy when applied to formalin-fixed paraffin-embedded (FFPE) tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.
Chromosomal translocations can cause cancer, often through the formation of fusion genes that code for an unnatural tyrosine kinase that promotes constitutive activation of a signaling pathway controlling cell proliferation and differentiation. For example, the diagnostic hallmark of chronic myelogenous leukemia (CML) is an oncogene fusion formed from a reciprocal translocation (t(9;22)(q34.1;q11.2)) between chromosomes 9 and 22 that results in an altered chromosome 22q known as the Philadelphia chromosome. Approximately 95% of all CML patients harbor the gene fusion, BCR-ABL, which is formed via a double stranded break (DSB) within both the Abelson oncogene 1 (ABL) on chromosome 9q, which codes for a non-receptor tyrosine kinase (ABL), and the breakpoint cluster region gene (BCR) on chromosome 22q. BCR-ABL encodes a constitutively active tyrosine kinase BCR-ABL responsible for the uncontrolled proliferation associated with chronic myelogenous leukemia. The identification of these translocation events and/or associated fusion genes in clinical samples is critical to ensure the appropriate treatment for patients where the drug and related course of therapy target an activated fusion kinase. Clinical detection of complex chromosomal rearrangements is often conducted using fluorescence in situ hybridization (FISH). The FISH analysis, though effective, offers relatively poor sensitivity while being expensive, time-consuming and technically challenging to perform. Here we have developed and validated a new general platform for creating assays against complex chromosomal rearrangements, including both reciprocal and non-reciprocal translocations. It utilizes droplet digital PCR (ddPCR) technology in lieu of FISH to quantify the rearrangement of proto-oncogenes that undergo rearrangement as part of the translocation event. The platform is applied to the creation of two new assays of potential clinical use in cancer diagnostics or theranostics. The first provides a reliable and sensitive measure of DSBs within the major breakpoint region of BCR (M-BCR), permitting initial diagnosis of CML through unequivocal detection of the BCR-ABL fusion gene to a frequency of 0.25%. The second provides for the highly sensitive detection of DSBs in the anaplastic lymphoma kinase (ALK) gene that result in a non-reciprocal (inversion) translocation (inv(2)(p21;p23)) associated with an ALK-positive non-small cell lung cancer (NSCLC).
Biological reagents that recognize target molecules with high affinity and specificity are widely used as capture agents, diagnostic reagents, and therapeutics. Through their ability to adopt structures that confer binding affinity for a target, aptamers represent one major class of such reagents. However, their use is limited by the general inability of current selection methods to reliably discover high-quality aptamers. Inefficiencies in their selection are due in part to a lack of fundamental understanding of the mechanisms underpinning each step in the screening process.This thesis reports on a series of studies conducted to define the factors and mechanisms currently limiting aptamer selections. That knowledge is then used to create highly effective strategies and technologies for ameliorating each limitation affecting their selection. The resulting collection of improvements is integrated into a novel selection workflow termed “Hi-Fi SELEX”. Those improvements include i) application of a novel “competent library” that eliminates fixed-region interference effects during selection, ii) development of effective chemistries to optimally retain desirable library members, iii) invention of simple methods to accurately quantify retained library diversity and mean binding affinity after each selection round, and iv) development of emulsion PCR methods to eliminate generation of amplification artifacts and v) achieve stoichiometric recovery of the desired single-stranded aptamer library. The resulting discovery platform greatly improves the reliability and speed in which useful panels of lead aptamers against several clinically-relevant targets are discovered.Following initial selection of candidate aptamers based on binding affinity, further screening is typically required, in part to ensure target-specific binding – a performance need shared by antibodies selected against specific targets. However, moderate to high-throughput methods to efficiently screen panels of candidates for binding specificity are lacking. A new technology enabling label-free specificity screening of antibody or aptamer populations at suitable throughputs was therefore established at the proof-of-concept level. The novel microfluidic SPRi arrays described permit multiplexed detection of lead candidates by quantifying both equilibrium binding constants and binding kinetics for each interaction in an element-addressable fashion. The technology offers the ability to independently interrogate candidate affinity reagents and then recover those samples for downstream analysis.
Isoelectric chromatofocusing (ICF), a mode of chromatography by which proteins are separated based on changes in their charge with pH, is widely used at analytical scales, but its use in bio-product manufacturing has been limited. This is partly due to poor knowledge about operating ICF at scale, lack of understanding of its elution mechanisms, and the use of complex, costly buffers. Work presented in this thesis focuses on advancing ICF at both analytical and preparative scales.A method for generating pH gradients in ICF is developed using simple low-molecular-weight buffers. On anion and cation exchange media, linear gradients spanning more than six pH units are generated through isocratic or gradient interchange of loading and elution phases. The buffers used are selected to satisfy cost constraints and for compatibility with detection by UV absorption at 280 nm and mass spectrometry.A new surface-reaction/chemical-equilibria model is derived and solved by computer-aided simulations to predict pH and ionic strength profiles generated on anion and cation exchange columns. The model can be used for in silico design of custom-shaped elution profiles to improve separation performance. The method is used to achieve high purity and process throughput of a desired isoform of recombinant N-lobe of human transferrin produced by Pichia pastoris using custom isocratic ICF on preparative media. Gradient sculpting methods are used to enhance ICF as the first dimension in a multidimensional separation platform used for the detection and analysis of O-linked N-acetylglucosamine modified proteins within the proteome of differentiated C2C12 mouse myoblast cells.Finally, a model of protein transport and binding in ICF is developed and used to show that elution is not dictated solely by a protein’s isoelectric point (pI), but is instead multi-modal in nature with Donnan equilibria, ion-exchange, and ion-displacement effects at work. The model predicts how simultaneous modulation of ionic strength and pH during elution can greatly improve the separation of proteins with similar pI’s; elution characteristics including retention time, peak width and resolution can likewise be improved. By coupling mathematical relationships describing these elution mechanisms to the solution of the continuity equation, protein elution times are accurately predicted.
Cancer is characterized as a genetic disease associated with acquired somaticmutations, a majority of which consist of only a single base change and are commonlyreferred to as somatic point mutations (SPM). Real-time quantitative polymerase-chainreaction (qPCR) techniques using allele specific (AS) probes or primers are widely used ingenotyping assays to detect commonly known single nucleotide polymorphisms (SNP), andalso have the potential to detect SPMs, provided the required analytical sensitivity andspecificity can be realized. One strategy to establish the necessary performance is tointroduce nucleotide analogs such as Locked Nucleic Acids (LNAs) into AS probes orprimers; however the successful design requires a fundamental understanding of both thethermodynamics and kinetics of LNA-DNA heteroduplexes. Melting thermodynamic studiesof DNA duplexes and LNA-DNA heteroduplexes were therefore carried out using bothultraviolet (UV) spectroscopy and differential scanning calorimetry (DSC) to quantify thethermodynamics (ΔH⁰, ΔS⁰, ΔCp and Tm) associated with the helix-to-coil transition. Datacollected on DNA duplexes and DNA-LNA heteroduplexes were used to introduceimprovements in the “unified” nearest-neighbor model, and for the development of a newmodel, referred to as the Single Base Thermodynamic (SBT) model that accurately predictsthe Tm for the melting of LNA-DNA heteroduplexes.The SBT model was extended and applied to PCR conditions to design LNA-bearingAS probes for qPCR assays to detect the clinically important SPMs KIT c.1799t>a (D816V)and JAK2 c.1849g>t (V617F), and were found to significantly outperform standard ASprobes containing only DNA. The interaction of Taq polymerase with heteroduplexesformed between an LNA-bearing primer and a target template were also studied and results used to generate general rules for designing LNA-bearing AS primers capable of unequivocaldetection of a rare mutant allele bearing a SPM. The method was then extended to allowqPCR detection by Plexor™ technology and applied to create an AS primer directed againstthe JAK2 V617F SPM that can detect one mutation in a background of more than 100,000copies of the wild-type allele and which is now used by the Cancer Genetics Laboratory ofthe British Columbia Cancer Agency (BCCA) to analyze patient samples.
Controlled-shear affinity filtration (CSAF) is a novel integrated bioprocessing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The rotor provides a tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation allowing for a uniform filtrate flux that maximizes membrane column performance. However, the fundamental hydrodynamics and mass transfer kinetics within the CSAF device are poorly understood and, as a result, the industrial applicability of the technology is limited. A computational fluid dynamic (CFD) model is developed that describes the rotor chamber hydrodynamics of the CSAF device. Once evaluated the model is used to show that a rotor of fixed angle does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress threshold across the membrane surface, compromising the scale-up of the technology. The CFD model is then used to model design improvements that result in an in silico design of a preparative CSAF device capable of processing industrial feedstocks.To describe mass transfer in stacked-membrane chromatography a novel zonal rate model (ZRM) is presented that improves on existing hold-up volume models. The ZRM radially partitions the membrane stack and external hold-up volumes to better capture non-uniform flow distribution effects. Global fitting of model parameters is first used under non-retention conditions to build and evaluate the appropriate form of the ZRM. Through its careful accounting of transport non-idealities within and external to the membrane stack, the ZRM is then shown to provide, under protein retention conditions, a useful framework for characterizing putative protein binding models, for predicting breakthrough curves and complex elution behavior, and for simulating and scaling separations using membrane chromatography.By elucidating the intrinsic physical processes ongoing in CSAF the mathematical models presented in this thesis represent essential theoretical tools for the further development of the technology; a technology which has the potential to increase productivity and decrease costs in the downstream processing of biopharmaceuticals.
No abstract available.
Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Biotechnology companies are now well skilled in the technologies and operations needed to manufacture biologic drugs safely for the treatment of major diseases. Those technologies have enabled development of highly efficient and cost-effective platforms for downstream processing of monoclonal antibody (mAb) based drugs. But translating those advances to create cost-effective DSP platforms for non-mAb protein therapeutics having relatively low annual production rates, often termed “orphan” drugs, has proven difficult. A key driver of the efficiency of mAb DSP platforms is the use of protein A affinity chromatography to capture and purify the product directly from clarified culture supernatants. Unfortunately, effective ligands for affinity capture of non-mAb biologics are generally not available. But this could change through the development of a technology that rapidly discovers and validates cost-effective affinity ligands against non-antibody protein targets. This project describes the development of a new technology pipeline to accelerate the discovery, optimization, and validation of affinity chromatography media that is specifically tailored to provide for robust economical capture of non-mAb biologic drugs from complex cell cultures. It is based on the use of DNA aptamers as affinity ligands discovered using an advanced aptamer screening technology we call High-Fidelity Systematic Evolution of Ligand by Exponential Enrichment (Hi-Fi SELEX). The refined, truly robust Hi-Fi SELEX technology described in this thesis greatly improves upon a proof-of-concept version of that method we recently described.This second-generation Hi-Fi SELEX method was used to successfully select high-affinity ligands against two non-mAb target proteins, human complement Factor D and human mesothelin. Anti-Factor D (aFD-30) aptamer against Factor D was then used as ligands in preparative affinity chromatography columns. The chemically modified aFD-30 with 3’ inverted dT nucleotide cap was immobilized on preparative affinity chromatography matrix for the capture and purification of Factor D from CHO cell supernatant. Standard column performance data were collected, including static and dynamic binding capacities, purities, concentration factors, and yields, which showed excellent separation performance. These results therefore demonstrate the potential of the proposed technology for custom design and validation of preparative chromatography media that can benefit the growing orphan drugs market by reducing manufacturing costs.
Biological reagents that bind a target selectively and with high affinity are widely used as recognition molecules within diagnostic assays and as therapeutics, among other applications. By leveraging their Watson-Crick base pairing ability, short DNA oligonucelotides represent one class of such biological agents that is particularly well suited to analyzing specific elements of the human genome. Such analyses are routinely used by clinics to detect and manage disease, and those analyses are increasingly providing the richer data content and improved performance necessary for effective clinical decision-making by employing chemically modified nucleic acids. To date, the use of these unnatural nucleotides has largely been achieved empirically, but their growing use is motivating the development of new tools and guidelines that accelerate and improve their implementation in novel assays. This thesis describes how two experimental methods may be tailored to accurately measure the melting thermodynamics of short duplex DNA containing chemical modifications – specifically locked nucleic acids (LNAs) – and then reports on a study that used those methods to measure the thermal stabilities of a large panel of DNA duplexes containing LNA substitutions in one or both strands. Those data and insights gleaned from them are used to extend a molecular thermodynamic model, the “Single Base Thermodynamic” (SBT) model, to enable accurate predictions of the melting thermodynamics of short B-form DNA duplexes containing i) LNA:LNA base pair and/or ii) oppositely oriented LNA:DNA base pair structures. It is the only thermodynamic model with this ability, and its value is demonstrated through its use to guide the development of a entirely new type of quantitative real-time PCR based diagnostic assay – in this case directed against clinically relevant BRAFV600 mutations in cancer – that improves upon commercially available assays by bettering their throughput and limit of detection.