Profiling Hinge Plasticity in Intact Monoclonal Antibodies for Antigen Recognition

Area of Science:

• Biochemistry
• Structural Biology
• Biotherapeutics

Background:

• Monoclonal antibodies (mAbs) are crucial therapeutics, but their physicochemical properties can impact efficacy.
• Structural characterization of mAbs is essential for predicting and improving therapeutic variants.
• Glycosylation in mAbs hinders traditional isotope labeling for Nuclear Magnetic Resonance Spectroscopy (NMR) studies.

Purpose of the Study:

• To develop and apply an NMR-based structural fingerprinting methodology for intact therapeutic mAbs at natural abundance.
• To investigate the structural dynamics of the mAb hinge region in solution and its role in antigen binding.

Main Methods:

• Utilized NMR-based structural fingerprinting on three therapeutic mAbs without isotope labeling.
• Employed a peptide-based assignment methodology for intact proteins (approx. 150 kDa).
• Compared mAb fingerprints with Fc-fusion proteins to assess robustness.

Main Results:

• Successfully generated structural fingerprints for therapeutic mAbs at natural abundance.
• Identified the flexible hinge segment of mAbs in solution using the peptide-based assignment approach.
• Demonstrated hinge plasticity through fingerprint changes upon interaction with cognate and non-cognate antigens.
• Confirmed the robustness of the methodology by comparing with Fc-fusion protein fingerprints.

Conclusions:

• The developed NMR methodology enables structural characterization of intact therapeutic mAbs without isotope labeling.
• The study highlights the significant role of the flexible hinge region in antibody-antigen interactions, beyond the Complementarity-Determining Regions (CDRs).
• This approach provides insights into the dynamic basis of antibody function in solution, crucial for biotherapeutic development.