THE LES SHEMILT LECTURESHIP
Monoclonal antibodies (mAbs) are the dominant class of biologic therapeutics, comprising nearly half of all therapeutic proteins in current development pipelines, while DNA aptamers are one of the fastest growing classes of diagnostic and therapeutic agents. I will describe novel and complementary platforms for high-throughput screening of these two classes of molecules and then demonstrate their potential to improve discovery of lead therapeutic and diagnostic compounds. The first is a microfluidic platform co-developed in our laboratory that allows for functional screening and selection of mAbs secreted by single plasma cells, as well as light and heavy chain amplification, isolation and sequencing on a single highly integrated device. By compartmentalizing single cells in microfluidic chambers over 1000X smaller in volume than conventional micro-titre wells, each isolated cell can secrete its unique antibody at concentrations detectable by fluorescence microscopy. Here this concept is linked to an on-chip fluorescence-based bead assay and RT-PCR to measure antibody-antigen binding kinetics/affinity and to determine the sequence of each candidate mAb in a highly multiplexed and rapid manner when applied to screening of primary antibody-producing plasma cells. As part of this platform, I will describe a low-pressure bead packing technique for the robust integration of high-performance chromatography columns in microfluidic devices made by multilayer soft lithography. A novel column geometry is used to achieve rapid packing of multiple high-quality columns that may be applied to achieve multiplexed clean-up of RT-PCR products. I will then describe a second platform, High-Fidelity SELEX (Hi-Fi SELEX), that both accelerates and improves selection of DNA aptamers by ameliorating several limitations of current methods used for aptamer discovery. Improvements to aptamer selections will first be demonstrated using human a-thrombin as a benchmark target. Results for three clinically relevant targets (human factors IXa, X and D) subjected to Hi-Fi SELEX will then be described. For each, rapid enrichment of ssDNA aptamers offering an order-nM mean dissociation constant (Kd) is achieved within at most 3 selection rounds. Finally, results for a promising consensus sequence for treating atypical hemolytic–uremic syndrome (aHUS) through suppression of factor D-mediated complement activation will be presented.
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