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Proteolysis-targeting chimeras (PROTACs) represent a powerful therapeutic modality for selective protein degradation but often suffer from poor pharmacokinetics and limited tumor-targeting. To overcome these constraints, we developed albumin-binding BRD4-degrading PROTACs (Alb-TACs) with esterase-cleavable maleimide linkers that hitchhike endogenous albumin and enable esterase-responsive BRD4 degradation in tumors.

Alb-TACs were synthesized by conjugating two esterase-cleavable maleimide linkers, bicyclononyne-polyethylene glycol-maleimide (BCN-PEG₂-Mal) or N-(2-aminoethyl)maleimide (AE-Mal), to BRD4-degrading PROTAC (ARV-771), resulting in Alb-TAC#1 and Alb-TAC#2, with distinct albumin- and esterase-binding properties. To select effective Alb-TAC, the binding ability to albumin and esterase-specific cleavage of Alb-TACs were carefully assayed using MALDI-TOF, PAGE, and time-course HPLC. Furthermore, the tumor-targeting efficacy of Alb-TACs was assessed by fluorescence imaging in CT26 tumor-bearing BALB/c mice. Next, we investigated the BRD4 degrading efficiency of Alb-TAC in a cell culture system and in CT26 tumor-bearing mice. Finally, the immunogenic cell death (ICD) and reprogrammed immune cells of Alb-TAC-treated tumors were carefully characterized.

Alb-TAC#2 containing the AE-Mal linker exhibited rapid albumin binding, accelerated esterase-responsive activation, and enhanced tumor accumulation compared to ARV-771 and Alb-TAC#1 due to its flexible chemical structure. In the CT26 cell culture system, Alb-TAC#2 efficiently degraded BRD4, resulting in BRD4-deficient cell death. Furthermore, in CT26 tumor-bearing mice, Alb-TAC#2 achieved extensive apoptosis through robust BRD4 degradation, leading to marked downregulation of c-Myc, Bcl-2, and PD-L1. Moreover, Alb-TAC#2 induced hallmarks of ICD (elevated surface CRT, extracellular ATP, and HMGB1) and reprogrammed the tumor microenvironment by enhancing CD8⁺ T cell infiltration, promoting dendritic cell maturation, and reducing regulatory T cell function.

This esterase-responsive albumin-binding PROTAC design could overcome pharmacokinetic barriers of conventional BRD4-targeting PROTACs by enhancing tumor-specific delivery and esterase-responsive BRD4 degradation in solid tumors. In summary, esterase-responsive albumin-binding PROTAC is proven as a promising strategy that effectively modulates the pharmacokinetics and therapeutic performance of PROTACs for cancer immunotherapy.