Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

Phage display technology is a common approach for discovery of therapeutic antibodies. Drug candidates are typically isolated in two steps: First, a pool of antibodies is enriched through consecutive rounds of selection on a target antigen, and then individual clones are characterized in a screening procedure. When whole cells are used as targets, as in phenotypic discovery, the output phage pool typically contains thousands of antibodies, binding, in theory, hundreds of different cell surface receptors. Clonal expansion throughout the phage display enrichment process is affected by multiple factors resulting in extremely complex output phage pools where a few antibodies are highly abundant and the majority is very rare. This is a huge challenge in the screening where only a fraction of the antibodies can be tested using a conventional binding analysis, identifying mainly the most abundant clones typically binding only one or a few targets. As the expected number of antibodies and specificities in the pool is much higher, complementing methods, to reach deeper into the pool, are required, called deep mining methods. In this study, four deep mining methods were evaluated: 1) isolation of rare sub-pools of specific antibodies through selection on recombinant proteins predicted to be expressed on the target cells, 2) isolation of a sub-pool enriched for antibodies of unknown specificities through depletion of the primary phage pool on recombinant proteins corresponding to receptors known to generate many binders, 3) isolation of a sub-pool enriched for antibodies through selection on cells blocked with antibodies dominating the primary phage pool, and 4) next-generation sequencing-based analysis of isolated antibody pools followed by antibody gene synthesis and production of rare but enriched clones. We demonstrate that antibodies binding new targets and epitopes, not discovered through screening alone, can be discovered using described deep mining methods. Overall, we demonstrate the complexity of phage pools generated through selection on cells and show that a combination of conventional screening and deep mining methods are needed to fully utilize such pools. Deep mining will be important in future phenotypic antibody drug discovery efforts to increase the diversity of identified antibodies and targets.