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

Area of Science:

• Immunohistochemistry
• Molecular Biology
• Protein Chemistry

Background:

• The PATHWAY anti-HER2/neu (4B5) antibody is crucial for identifying patients eligible for anti-HER2 targeted therapy.
• Previous studies indicated potential cross-reactivity of the 4B5 antibody with HER4 and ZSCAN18 under experimental conditions.

Purpose of the Study:

• To fully characterize the epitope and binding properties of the 4B5 antibody.
• To identify the off-target proteins responsible for observed cytoplasmic and nuclear reactivity in non-breast tissues.
• To define the binding characteristics and detection limits for off-target interactions.

Main Methods:

• Surface plasmon resonance (SPR)
• Automated capillary electrophoresis (ACE)
• Immunohistochemistry (IHC)
• Protein purification
• Mass spectrometry
• In-silico docking analysis

Main Results:

• Key amino acids involved in 4B5 antibody recognition were identified.
• The 4B5 antibody was found to cross-react with aldo-keto reductase family 1 members B1, B10, and B15 (AKR1B1, AKR1B10, AKR1B15).
• The binding characteristics and limits of detection for these off-target interactions were defined.
• In-silico analysis revealed paratope plasticity enabling interaction with AKR1B family proteins due to complementarity-determining region loop flexibility.

Conclusions:

• The 4B5 antibody exhibits off-target binding to AKR1B family proteins, contributing to cytoplasmic and nuclear IHC staining.
• Understanding these off-target interactions is essential for accurate interpretation of IHC results and patient selection for targeted therapies.
• The characterized epitope and binding properties provide a foundation for improving antibody specificity or developing alternative diagnostic tools.