Abstract 1161: A systematic evaluation of off-target binding in antibody-based biologics during clinical and preclinical development

Abstract Background: Specificity profiling is a requirement for monoclonal antibodies (MAbs) and antibody-based biotherapeutics such as CAR-T prior to initiating human trials. Traditional approaches to assess MAb specificity, primarily tissue cross-reactivity (TCR) studies, have been unreliable and have resulted in undetected off-target binding. Cell-based protein arrays represent an alternative and improved assessment tool for MAb specificity that have been recommended in recent FDA guidance. Methods: We developed a cell-based protein array, the Membrane Proteome Array (MPA), to assess binding across the full human membrane proteome. The MPA encompasses ∼6, 000 membrane proteins, each individually expressed in their native structural configuration within unfixed cells. The MPA enables quantitative and high sensitivity detection using flow cytometry. Here, we used the MPA to estimate the frequency of off-target binding by candidate antibody therapeutics. We conducted a retrospective analysis of 250 customer preclinical antibodies screened on the MPA and produced and screened 83 biosimilars of FDA-approved and clinical-stage therapeutic antibodies. Results: Retrospective analysis of customer antibodies that were screened using the MPA indicates a surprisingly high off-target rate, with 33% of lead candidates displaying off-target binding. Moreover, about 20% of therapeutic MAbs in clinical development and currently on the market displayed off-target binding. To directly compare cell-based protein arrays to TCR studies, we compared our own MPA data for therapeutic MAbs to publicly available TCR data and in several cases identified discrepancies in off-target binding results. Conclusion: Overall, the presented case studies and off-target rates at the different phases of drug approval suggest that off-target binding is a major cause of adverse events and drug attrition that could be ameliorated with better specificity screening. Citation Format: Diana Norden, Carmen Navia, Tabb Sullivan, Alexander Hobby, Benjamin Doranz. A systematic evaluation of off-target binding in antibody-based biologics during clinical and preclinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1161.