Intracellular pharmacology and trafficking to support sequential activation of SynchroLINK T2X antibody-drug conjugates in cancer.

e15045 Background: Clinical performance of antibody–drug conjugates (ADCs) is often constrained by rapid payload release, Cmax-driven toxicity, and limited therapeutic index. SynchroLINK T2X ADCs are designed to enable sequential intracellular activation, potentially supporting sustained intracellular exposure while reducing non-specific toxicity. We evaluated the intracellular pharmacology of T2X ADCs to assess whether this activation paradigm produces exposure–response characteristics relevant to clinical translation. Methods: T2X ADCs were evaluated in cancer cell models expressing relevant surface targets using time-dependent cytotoxicity assays designed to model transient and sustained exposure conditions. Washout experiments were performed to assess persistence of intracellular pharmacologic activity following limited extracellular exposure. Confocal imaging and colocalization analyses were used to characterize subcellular distribution following internalization. Pharmacologic perturbation of intracellular transport and lysosomal function was employed to probe dependencies relevant to sequential activation. Results: T2X ADCs exhibited time-dependent cytotoxicity profiles distinct from conventional ADC comparators, with preserved potency following transient exposure and washout. Intracellular tracking demonstrated efficient internalization and predominant localization within perinuclear, membrane-associated compartments consistent with regulated intracellular processing rather than rapid diffusional payload release. Disruption of intracellular trafficking pathways attenuated cytotoxic activity, indicating dependence on controlled intracellular transport for functional activation. These findings support sustained intracellular pharmacologic activity despite limited extracellular exposure. Conclusions: Intracellular pharmacology and trafficking studies support a sequential activation mechanism for SynchroLINK T2X ADCs in cancer that decouples cytotoxic activity from short-duration extracellular exposure. This controlled intracellular behavior provides a translational rationale for improved therapeutic index and supports further clinical development of precision activation strategies for ADC-based cancer therapies.