Trifunctional Pyrrolidine-Carbamate Self-Immolative Spacers Decouple Enzymatic Activation from Drug Release in Antibody−Drug Conjugates

Antibody−drug conjugates (ADCs) typically rely on enzymatically cleavable linker−payload systems, in which linker cleavage directly triggers drug release. Here, we report a new class of trifunctional pyrrolidine-carbamate self-immolative (SI) spacers designed to decouple the enzymatic activation from the rate-determining payload release step. The SI spacers were temporarily masked with the Gly−Arg−PABC moiety, a deliberately promiscuous protease substrate aimed at preventing spacer degradation during ADC assembly. Upon proteolytic cleavage, an active pyrrolidine intermediate remained attached to the mAb and subsequently released the payload through an enzyme-independent cyclative carbamate cleavage. Two structurally related pyrrolidine spacers (A and B) were synthesized and incorporated into camptothecin- and DXd-based linker−payload modules, which were successfully conjugated to model mAbs. Mass spectrometry analyses confirmed the proposed two-step activation mechanism and demonstrated that drug release kinetics can be modulated through spacer design without altering the enzymatic activation process. The tertiary amine-containing spacer B led to improved ADC hydrophilicity and conjugate homogeneity while maintaining rapid degradation. Trastuzumab−DXd conjugates retained in vitro antiproliferative activity, supporting the feasibility of this linker−spacer architecture. These results establish trifunctional pyrrolidine-carbamate spacers as a versatile platform for the controlled release of alcohol-containing payloads and provide a foundation for independently tuning enzymatic activation and chemical drug release in future ADC designs.