Enabling the synthesis of multi-payload thio-antibody conjugates through the use of pyridazinediones,

In recent years, antibody-drug conjugates (ADCs) have emerged as a very promising class of targeted therapeutics, but ADC candidates still face issues such as dose-limiting toxicity and drug resistance. It has become increasingly clear that whilst there are general principles for what constitutes an effective ADC, individual ADCs require bespoke optimisation. The optimal drug-to-antibody ratio (DAR) may differ for each drug, antibody and target combination. Most recently, the use of multi-drug bearing ADCs to off-set drug resistance has aroused interest. In view of this, the modular construction of antibody conjugates with different DARs/drug classes is key to enabling the next generation of ADCs. One of the leading antibody scaffolds for making ADCs is antibodies with engineered cysteine residues (thio-antibodies). Typically, it is only the engineered site that is reacted when making ADCs from thio-antibodies, inherently limiting the potential of such conjugates. This work focuses on the development of a platform for the modular and site-selective synthesis of thio-trastuzumab mutant conjugates with defined payload-to-antibody ratios (PARs) of 1, 2, 3, 4, 5, 6, 7 and 8 by modifying engineered and native cysteines (note: in this manuscript the term payload refers to fluorophores or other functional small-molecule entities attached to an antibody). The framework to achieve this is based on using only three key molecules: a diBr-pyridazinedione (PD) bearing a strained alkyne, an azide-functionalised bisPD and a functionalised azido-aniline entity. This approach, combined with the use of clickable payloads, enables the synthesis of the full repertoire of PARs 1-8, and the ability to attach up to three different payloads, in different ratios, to thio-trastuzumab mutants. The distinct PARs and ratios of payloads can be tuned by changing the reagents used and/or the order of reagents used in combination with various clickable payload reagents. Finally, various tri-payload loaded thio-antibody conjugates were appraised