FlexTORCH: An Improved Flexible Fluorophore–Linker–Quencher Molecule Enlightening ADC Research

Antibody-drug conjugates (ADCs) are an emerging class of molecules for cancer therapy. An ADC consists of an antibody that is attached to a toxic payload via a linker molecule. Once the ADC is internalized into the cancer cell, the payload is released inside the cell, which leads to tumor cell death. Most approved ADC molecules make use of enzymatically cleavable linker structures. The kinetics of antibody internalization, linker cleavage, and payload release are evident for the mode of action of ADCs in vitro and in vivo. We have previously described the generation of the tool molecule TORCH (Turn On after Release by Cathepsin) for studying ADC kinetics by analyzing increasing fluorescence. The molecular TORCH is a fluorophore-quencher molecule that is separated by a valin-citrullin (VC) linker. The VC linker is cleaved by the protease cathepsin B. We previously demonstrated the in vitro proof of principle with the molecular TORCH. These studies strongly facilitated ADC research and the analysis of internalization and release kinetics. Here, we show an improved design of the TORCH molecule, also named flexTORCH, overcoming challenges in synthesis, conjugation, and flexibility of design. The flexTORCH molecule enables modular and flexible assembling. For this, different TORCH linker-quencher modules and the fluorophore were equipped with functional groups for orthogonal click chemistry. This study shows the feasibility of flexTORCH synthesis, its stepwise conjugation to trastuzumab, and the <i>in vitro</i> proof of principle. For showcasing the flexibility of the flexTORCH, four different constructs were produced, including VC-PABC (para-aminobenzyl carbamate), ß-glucuronide-PABC, AAN-PABC, and AAN linker that represent recognition patterns for cathepsin B, ß-glucuronidase, and legumain.