Assessment of ADC Higher Order Structure Through 2D NMR Analysis

Area of Science:

• Biochemistry
• Biophysics
• Chemical Biology

Background:

• Antibody-drug conjugates (ADCs) are vital chemotherapeutics.
• Understanding ADC physical properties is key to developing superior drugs.
• Biomolecular NMR shows potential for characterizing antibody structure in ADCs.

Purpose of the Study:

• To assess the impact of drug conjugation on antibody higher-order structure using NMR.
• To investigate the effects of varying drug-antibody ratios (DAR) on ADC structure.
• To explore the utility of 2D NMR for ADC characterization.

Main Methods:

• Applied 2D NMR techniques to trastuzumab and its conjugates (T-MMAE, T-DXd) at DAR 2, 4, and 8.
• Analyzed NMR spectra of trastuzumab alone and at different drug conjugation levels.
• Compared spectra to assess changes in antibody higher-order structure.

Main Results:

• Trastuzumab alone yielded high-quality NMR spectra.
• Low DAR conjugates showed spectra similar to free antibody, with new linker-payload peaks.
• Increasing DAR led to peak disappearance and global signal broadening, especially at high DAR (T-MMAE more affected than T-DXd).

Conclusions:

• Biomolecular NMR provides insights into ADC solution behavior.
• Drug conjugation, particularly at higher DAR, alters antibody higher-order structure.
• NMR is a promising tool for characterizing ADC physical properties.