Background Rigorous specificity analysis is critical for the development of antibody-based therapies, as even minimal off-target binding can lead to toxicity and clinical failures. Long believed to be exquisitely specific, recent preclinical data and our own work indicate that monoclonal antibodies (MAbs) frequently (~25%) display cross-reactivity. In many cases, off-target interactions occur with unrelated proteins that cannot be predicted by protein sequence homology. Cross-reactivity can lead to serious or even life-threatening consequences especially when MAbs are configured as bispecifics, antibody-drug conjugates (ADC) or CAR-T cell therapies, and IND applications for biotherapeutics require cross-reactivity assessment to prevent adverse events. Tissue cross-reactivity (TCR) studies have traditionally been used to screen for off-target binding, however, with poor predictive value for in vivo safety and toxicity.

Methods We developed the Membrane Proteome Array (MPA) platform to de-risk MAb-based therapeutics by testing for specificity across 6,000 human membrane proteins expressed in live cells. The MPA assesses binding interactions by high-throughput flow cytometry allowing for high sensitivity detection and rapid analysis. All targets identified on the MPA screen are validated by secondary titration analysis. In contrast to TCR studies, proteins in the MPA exist in their native conformations and are not altered by fixation.

Results Recently, we used the MPA to select a lead antibody candidate targeting the oncotherapeutic target Claudin 6 (CLDN6). Through MPA screening, we successfully identified a lead candidate devoid of cross-reactivity against other CLDN family members or any other membrane protein across the human genome. The MPA has also been used to rapidly evaluate the specificity of novel CAR-T cell therapies for cancer and autoimmunity.

Conclusions The MPA has been used for lead selection and data from the MPA has been accepted by the FDA as a part of IND applications for antibody-based therapies. The MPA is currently under consideration for being developed as an FDA-qualified Drug Development Tool.