Accelerated Antibody Evolution Under Functional Selection

About This Grant

PROJECT SUMMARY Antibodies have proven to be a powerful and effective therapeutic strategy, with successful drugs targeting a variety of pathways. New antibodies can be developed by animal inoculation or by ex-vivo methods such as phage-display. The former harnesses the power of somatic hypermutation that is a natural component of the mammalian immune system, while the latter circumvents the labor and cost associated with animal experimentation. Both approaches produce antibodies characterized by strong binding to the antigen target of interest. Strongly binding antibodies must then be further screened to determine their functional activity, namely their ability to either stimulate or block activity in a specific cell signaling receptor. Importantly, these approaches do not harness the power of evolution to optimize the functional characteristics of the antibody. The goal of this project is to develop an in vitro method to perform rapid antibody evolution, where selection is performed based on the function of the antibody rather than just the strength of antigen binding. Specifically, the project will aim to develop allosteric modulators for G protein-coupled receptors (GPCR), which are the largest family of membrane proteins in humans and broadly represented across nearly all physiological and pathophysiological processes. The project will leverage an engineered system in which a yeast plasmid replicates orthogonally from the rest of the yeast genome such that the plasmid is copied with a high error rate to produce rapid evolution while the rest of the yeast genome is replicated with low error rate to maintain the health of the organism. Antibody fragment sequences will be encoded on the rapidly mutating plasmid and secreted by the yeast. The yeast will be encapsulated in microfluidic double-emulsion droplets together with mammalian GPCR reporter cells. Antibody function will thus be quantified by fluorescent reporter, and droplets containing yeast with a desired functionality can be selected via droplet sorting by conventional fluorescence-activated cell sorting. After droplet demulsification, the selected yeast can be further expanded to continue the evolutionary process.