[Identifying protein epitopes recognized by monoclonal antibodies]

Area of Science:

• Immunology
• Protein Biochemistry
• Biotechnology

Background:

• Therapeutic monoclonal antibodies (mAbs) are crucial in treating various diseases.
• Precisely identifying the epitopes recognized by mAbs is essential for understanding their mechanism of action and for developing new antibody-based therapies.
• Programmed death receptor-1 (PD-1) is a key immune checkpoint target for cancer immunotherapy.

Purpose of the Study:

• To develop and validate a rapid and accurate method for identifying protein epitopes targeted by therapeutic monoclonal antibodies.
• To determine the specific epitopes of the anti-PD-1 antibody Pembrolizumab using the established method.

Main Methods:

• An alanine scanning strategy was employed, utilizing site-directed mutagenesis combined with a mammalian cell expression system.
• Established conditions for eukaryotic expression element amplification and cell transfection for antigen mutant expression.
• Co-expressed 150 programmed death receptor-1 (PD-1) protein mutants and assessed their binding affinity to Pembrolizumab.
• Integrated binding data with protein structure analysis to pinpoint antibody epitopes.

Main Results:

• Successfully established a rapid expression system for antigen mutants.
• Identified the binding capabilities of 150 PD-1 mutants to the anti-PD-1 antibody Pembrolizumab.
• Determined the epitopes of Pembrolizumab, which showed high consistency with previously reported crystal structure-based epitopes.

Conclusions:

• The developed method is simple, accurate, and efficient for epitope mapping of therapeutic monoclonal antibodies.
• This technique provides a valuable tool for characterizing antibody-antigen interactions, particularly for immune checkpoint inhibitors like anti-PD-1 antibodies.
• The findings support the utility of this method for advancing antibody-based drug discovery and development.