Generating Recombinant Affinity Reagents by Phage- and Ribosome-Display

Considering the fields of therapeutics, biological sensing, and basic biology, new research tools are in high demand. Monoclonal antibodies have answered some of these demands. However, the cost and time required for antibody development decreases their potential. For these reasons we have looked to recombinant reagents that can be generated in tubes, expressed in bacteria, and renewed indefinitely. They can be engineered to tackle specific problems, include tags and modifications, and have their sequences synthesized anywhere in the world. These reagents are becoming of higher importance as chemical drug libraries are reaching exhaustion, early disease diagnosis is crucial for patients, and criticism of animal research is coming to a head. In this thesis, we have engineered scFv antibodies to detect biomarker proteins released into bodily fluids indicative of retinal injury caused by laser exposure. One of these antibodies generated by phage-display, detected a biomarker protein in animal retinal lysates. We attempted to improve the binding through affinity maturation, however we were plagued by a propensity to isolate weak binding but high expressing clones. To make the antibody more useful, we dimerized the recognition epitopes through an Fc region to get an increase in sensitivity due to avidity. Considering the shortcomings experienced attempting to increase the utility of recombinant antibodies, we developed a novel process to obtain high quality reagents in a high-throughput manner. This process has combined two protein display technologies, phage and ribosome display, in a novel fashion that utilizes advantages of both. We have dubbed the technology, Primer Extension for Selection Recovery or PExSR. In practice, PExSR resulted in reagents with equilibrium dissociation constant (KD) in the picomolar range. These were 100-fold better than we have seen in the past using solely phage-display, and took a third of the time. As we can also multiplex the procedure to include many targets, we anticipate this method to be attractive to industry because the initial libraries are more simple to generate, have large starting libraries (1x10^13), and quickly result in reagents that have specificity and affinity for various biological applications.