Paratope plasticity determines anti-HER2 (4B5) antibody specificity

The PATHWAY anti-HER2/neu (4B5) Rabbit Monoclonal Primary Antibody immunohistochemistry (IHC) assay is used to identify patients eligible for anti-HER2 targeted therapy. Previous work has demonstrated that the 4B5 antibody can react with the related family member HER4, and the nuclear protein ZSCAN18, under experimental conditions. Here, we used surface plasmon resonance, automated capillary electrophoresis (ACE), and IHC to fully define the 4B5 epitope and binding characteristics. The key amino acids that contribute to antibody recognition were identified and characterized with the limits of detection determined for HER4 and ZSCAN18. Additionally, the protein target responsible for cytoplasmic and nuclear reactivity observed by 4B5 in non-breast tissue types, that is not attributable to either HER2, HER4, or ZSCAN18, was identified. By means of protein purification, mass spectrometry, ACE analysis and IHC, 4B5 was found to react with the cytosolic aldo-keto reductase family 1 members B1, B10 and B15 (AKR1B1, AKR1B10, AKR1B15). The binding characteristics and limits of detection for these off-target species were defined. In-silico docking analysis of computationally folded 4B5 paratope regions identified key differences in the 4B5 paratope interaction between a peptide containing the HER2 epitope versus that of a partially homologous peptide sequence common to the AKR1 family. Flexibility of the complementarity-determining region loops in 4B5 imparts paratope plasticity that enables interaction with AKR1B family proteins. This antibody off-target recognition can result in cytoplasmic and nuclear IHC staining.