Assessment of ADC Higher Order Structure Through 2D NMR Analysis.

Antibody-drug conjugates (ADCs) represent a growing class of important chemotherapeutic molecules. Our understanding of the physical properties of the antibody, linker, and payload is still quite limited, but a better understanding may lead to superior ADCs. Biomolecular NMR has shown promise in the characterization of antibody higher-order structure, suggesting that the same should be true for ADCs. We applied 2D NMR techniques to trastuzumab alone and to trastuzumab conjugated to MMAE and DXd linker-payloads at drug-antibody ratios (DAR) 2, 4, and 8 to assess the effects of drug conjugation on antibody higher-order structure. Trastuzumab alone generated high-quality NMR spectra under a variety of temperatures and concentrations. Spectra of low DAR species were remarkably similar both to each other and to the free antibody, except for notable new peaks in the spectra from the linker-payloads. Increasing DAR resulted in the disappearance of many well-dispersed peaks; at the highest DAR, both T-MMAE and T-DXd showed a global broadening of signals, although this effect was more extreme in T-MMAE. These spectra demonstrate the promise of biomolecular NMR to provide a direct window into the solution behavior of ADCs.