Residue-Level Characterization of Antibody Binding Epitopes Using Carbene Chemical Footprinting

Area of Science:

• Biochemistry
• Immunology
• Drug Discovery

Background:

• Antibody binding epitope characterization is crucial for therapeutic drug discovery, influencing antibody pharmacology and pharmacokinetics.
• Understanding the precise binding site is essential for selecting and developing effective antibody-based therapeutics.

Purpose of the Study:

• To introduce a novel carbene chemical footprinting method coupled with mass spectrometry for high-resolution antibody epitope mapping.
• To demonstrate the application of this technique for characterizing epitopes of MICA and CTLA-4 antibodies.

Main Methods:

• Utilized photoactivated diazirine reagents for carbene chemical footprinting to label antibody binding interfaces.
• Employed mass spectrometry for identification of labeled residues at a single-residue level.
• Validated findings using X-ray crystallography and yeast surface display epitope mapping.

Main Results:

• Successfully mapped antibody binding epitopes at single-residue resolution.
• Provided complementary labeling information using two different diazirine reagents for structural refinement.
• Enabled structural understanding of competitive binding interactions and antibody selection.

Conclusions:

• Carbene footprinting offers a rapid, high-resolution method for epitope characterization in antibody discovery.
• This technique accelerates the drug discovery process by providing critical mechanistic insights into antibody function.
• Facilitates informed antibody selection and development for therapeutic applications.