RG-7598

Bifunctional trans-cyclooctene (bTCO) with a carbamate or carbonate at the allylic position and tetrazine provide a promising bioorthogonal click chemistry pair for the click-to-release approach, successfully employed in various biotechnological applications. Herein, we demonstrate a simple and straightforward method to synthesize C₂TCO, a symmetrical bTCO derivative with two hydroxyl groups at the allylic positions. The efficiently synthesized C₂TCO at first was selectively functionalized with a fluorophore (C₂TCO-FL), and the conjugate was labeled onto monoclonal antibodies (Ab-C₂TCO-FL). The fluorophore of Ab-C₂TCO-FL was easily removed from the antibody through the mild treatment of tetrazine, enabling multicycle fluorescent bioimaging. Next, an antibody-drug conjugate targeting PD-L1 was prepared using the linker based on C₂TCO. The cytotoxic payload was efficiently released from the antibody upon tetrazine treatment, which induced cellular cytotoxicity.

During the past decade, antibody-drug conjugates (ADCs) have emerged as new drugs in cancer therapy with 15 ADCs already approved such as Kadcyla, Enhertu, and Adcetris. ADCs contain a cytotoxic drug that is linked to an antibody, allowing for specific delivery of the warhead to tumor cells. Typically, the antibody targets a tumor-specific antigen expressed on the cell surface. After the internalization of ADCs into cells, the linker is often cleaved by enzymes in the lysosomal compartment of the cell, releasing the warhead and thereby allowing for its interaction with, for example, the DNA or the tubulin cytoskeleton, which finally leads to cell death. Consequently, binding, internalization, and drug release are key attributes for the efficacy of ADCs. Here, we describe a novel molecule named TORCH (Turn On after Release by CatHepsins) that contains a fluorescence quencher system that is separated by a cathepsin B-cleavable linker. When conjugated to an antibody, the TORCH molecule allows one to gain valuable insights on the internalization and drug release of ADCs. While we cannot exclude the influence of other factors such as receptor recycling, we have found that the receptor density is directly related to the amount of payload released intracellularly, meaning that the internalization per receptor is very similar for all investigated antibodies and cell lines.