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.

Systematic Development of Sandwich Immunoassays for the Plasma Secretome.

The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity-free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

Bead-Based and Multiplexed Immunoassays for Protein Profiling via Sequential Affinity Capture.

Antibody microarrays offer high-throughput immunoassays for multiplexed analyses of clinical samples. For such approaches, samples are either labeled in solution to enable a direct readout on the single binder assay format or detected by matched pairs of capture and detection antibodies in dual binder assay format, also known as sandwich assays. Aiming to benefit from the flexibility and capacity offered by single binder assay readout and the specificity and sensitivity of dual binder assays, we developed a multiplexed dual binder procedure that is based on a sequential, rather than combined, antigen binding. The method, entitled dual capture assay (DCA), is composed of an initial antigen capture by antibodies on beads, followed by labeling of captured protein targets on beads, combinatorial elution steps at high and low pH, and a readout using a secondary bead array. Compared to classical single binder assays, the described method demonstrated several advantages such as reduced contribution of off-target binding, lower noise levels, and improved correlation when comparing with clinical reference values. This procedure describes a novel and versatile immunoassay strategy for proteome profiling in body fluids.

Multiplexed protein profiling by sequential affinity capture.

Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.

The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas.

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.