A new approach methodology (NAM) for specificity testing: ISTAND qualification of the membrane proteome array to evaluate off-target binding of MAb-based therapies

Background: Scientifically validated New Approach Methodologies (NAMs) provide an opportunity to better predict drug safety in humans while also accelerating drug development and regulatory review. The FDA has established pathways to qualify NAMs as Drug Development Tools (DDTs) through various mechanisms that now include the Innovative Science and Technology Approaches for New Drugs (ISTAND) program. Methods: We developed the Membrane Proteome Array (MPA) platform for specificity testing of antibody-based therapeutics. The MPA assesses binding interactions across ~6,000 membrane proteins, each individually expressed in their native structural configuration within live or unfixed cells. The screen uses high-throughput flow cytometry, allowing for quantitative and high-sensitivity detection, and any identified targets are validated by titration analysis. The MPA is being qualified as a novel DDT through FDA's ISTAND program. Results: As part of our DDT qualification, we performed a comparison study of 35 antibody therapeutics evaluated for specificity using both our MPA and conventional tissue cross-reactivity (TCR) studies. We found that 17% of these drugs had an off-target that was not expected based on TCR results. Overall, the MPA and TCR results were consistent for only about half of the molecules tested. In numerous instances, TCR results were inconclusive based on technical variability, nonspecific or cytoplasmic staining, or false-positive staining. Such results highlight the difficulty of using TCR data for deciding actionable next steps. In contrast, off-targets from the MPA are de-risked by statistically quantifying relative affinity and binding levels, localizing binding to extracellular vs. intracellular epitopes, and determining tissue expression patterns in humans to assess the in vivo safety risk. Through the MPA qualification process, we have also quantified the sensitivity and reproducibility of the platform and implemented additional quality control procedures and data analyses. Conclusions: MPA data is already routinely used in regulatory applications for antibody-based biotherapeutics and may be used to replace or complement other cross-reactivity studies. The MPA is currently in the last stages of review for DDT qualification through FDA's ISTAND program.