Abstract
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.
Highlights
The efficacy of an antibody drug conjugates (ADCs) relies on numerous factors including characteristics of the ADC itself as well as the biologic features of the target and tumor cell
These variants enabled us to examine the premise that a weakened monovalent binding affinity, coupled with the avidity potential of bivalent mAbs, would confer preferential targeting of cells overexpressing the antigen and lead to reduced ADC toxicity on cells expressing a low level of antigen
This study describes a comprehensive regimen for pharmacological characterization and optimal selection of ADC candidates based on their intrinsic binding properties
Summary
The efficacy of an ADC relies on numerous factors including characteristics of the ADC itself as well as the biologic features of the target and tumor cell. The above studies, which dealt with non-conjugated antibodies as therapeutics, convey the importance of evaluating a wide affinity range and exploiting differential target density/avidity effects at the cell surface. Incorporated upfront in any procedure for screening antibodies for their potential as ADCs. Since high target density on the surface of a cancer cell has been shown to promote binding of low-affinity antibodies, it is apparent that weaker antibodies should not be de-prioritized in the early-phase ADC selection processes. As proof-of-principle, we applied our screening procedure to a rationally designed panel of Herceptin variants ranging from high to low affinity for Her2 These antibodies were ranked based on relative binding affinity to cells having high or low-Her densities and were classified as strong, moderate or weak binders. Strong binding Her antibodies with affinity KDs of ~1 nM or less exhibit high toxicities on both high- and low-Her cells, which may be disadvantageous in the context of very potent payload drugs such as PNU
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