Enabling the synthesis of multi-payload thio-antibody conjugates through the use of pyridazinediones, p-anisidine derivatives and various click chemistries

Abstract

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 in vitro, and were shown to bind and internalise selectively to HER2⁺ (BT-474) over HER2⁻ (MCF-7) cells with all payloads successfully delivered into target cells.