Binding and functional profiling of antibody mutants guides selection of optimal candidates as antibody drug conjugates

English version

Autoři: John C. Zwaagstra ^aff001; Traian Sulea ^aff001; Jason Baardsnes ^aff001; Stevo Radinovic ^aff001; Yuneivy Cepero-Donates ^aff001; Alma Robert ^aff001; Maureen D. O’Connor-McCourt ^aff002; Ilia A. Tikhomirov ^aff002; Maria Luz. Jaramillo ^aff001
Působiště autorů: Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, Canada ^aff001; Forbius, Montreal, Quebec, Canada ^aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0226593

Souhrn

An increasingly appreciated conundrum in the discovery of antibody drug conjugates (ADCs) is that an antibody that was selected primarily for strong binding to its cancer target may not serve as an optimal ADC. In this study, we performed mechanistic cell-based experiments to determine the correlation between antibody affinity, avidity, internalization and ADC efficacy. We used structure-guided design to assemble a panel of antibody mutants with predicted Her2 affinities ranging from higher to lower relative to the parent antibody, Herceptin. These antibodies were ranked for binding via SPR and via flow-cytometry on high-Her2 SKOV3 cells and low-Her2 MCF7 cells, the latter acting as a surrogate for low-Her2 normal cells. A subpanel of variants, representative of different Her2-binding affinities (2 strong, 2 moderate and 3 weak), were further screened via high-content imaging for internalization efficacies in high versus low-Her2 cells. Finally, these antibodies were evaluated in ADC cytotoxicity screening assays (using DM1 and MMAE secondary antibodies) and as antibody-drug conjugates (DM1 and PNU159682). Our results identified specific but weak Her2-binding variants as optimal candidates for developing DM1 and PNU ADCs since they exhibited high potencies (low to sub-nM) in high-Her2 SKOV3 cells and low toxicities in low-Her2 cells. The 2 strong-affinity variants were highly potent in SKOV3 cells but also showed significant toxicities in low-Her2 cells and therefore are predicted to be toxic in normal tissues. Our findings show that pharmacological profiling of an antibody library in multiple binding and functional assays allows for selection of optimal ADCs.

Klíčová slova:

Adenocarcinomas – Cell binding – Cell binding assay – Cytotoxicity – Flow cytometry – Fluorescence competition – Fluorescence imaging – Toxicity

Zdroje

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Binding and functional profiling of antibody mutants guides selection of optimal candidates as antibody drug conjugates

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