Antibody-Drug Conjugates (ADCs) are comprised of three distinct components, an antibody, a payload (usually a cytotoxic agent) and a linker (a synthetic chemical chain joining the antibody and payload). Payloads can be conjugated to antibodies in either a stochastic or site-specific manner, with stochastic conjugations usually accomplished through either amine groups of lysine residues or through thiol groups in disulfide bonds located in the hinge region of antibodies. In the latter case, an electrophilic Michael acceptor grouping (e.g., maleimide) of a linker-payload construct reacts with a nucleophilic thiol group of the antibody, forming the ADC. In order for this to occur, disulfide bonds need to be reduced to free thiols. However, knowing the concentration of reactive thiols at different stages during the reduction process can be challenging, and so a more accurate assessment of thiol concentration may allow greater control of the desired average Drug-Antibody Ratio (DAR). Currently, different spectrophotometric (i.e. Ellman’s reagent) and fluorometric assays are used to monitor thiol concentration, but these methods can be expensive, time consuming and inaccurate.

To address these issues, a rapid and efficient fluorescence-based method has been developed to allow the reduction of thiols to be monitored. The method is based on a fluorescein derivative produced via a four-step synthesis starting from commercially available fluorescein sodium salt. In its unreacted form, this novel probe exists in a lactone form. However, once it reacts with a free thiol group, it converts stoichiometrically via opening of the lactone ring to a fluorescent form, thus allowing quantitative determination of the concentration of thiol groups present in solution.

The three antibodies used to trial this procedure (i.e., Trastuzumab, Cetuximab and Rituximab) were reduced using TCEP over a 120 minute period. Aliquots of solution were collected at various time intervals, and incubated with the fluorescein probe for 5 minutes, monitoring fluorescence with a simple fluorimeter. Using this approach, it was possible to accurately assess thiol concentration at each time interval, and relate the overall reduction profile of each antibody to the concentration of reducing agent used. Payloads were then conjugated to the three antibodies at specific timepoints (selected through monitoring thiol reduction) in an effort to generate species with a defined DAR. Overall, the resulting ADCs were found to have average DAR profiles consistent with the measured number of thiols in the reduced antibodies.

When compared with other assays, the developed method gave results with improved reproducibility. Furthermore, the fluorescein probe was less complex and faster to use than other fluorometric kits, and was easy to synthesize, making it a useful new tool in the generation of ADCs.

Citation Format: Ilona Pysz, Paolo Andriollo, Paul J. Jackson, K M. Rahman, David E. Thurston. Development of a rapid and efficient methodology to quantitate thiol detection in antibody-drug conjugates (ADCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 217.