Synchronized CD73 blockade and STING activation via an ultrahigh-payload antibody-polymer conjugate for breast cancer immunotherapy

The enhanced antitumor immunity of stimulator of interferon genes (STING) agonists in breast cancer is hampered by unfavorable pharmacokinetics, targeting inefficiency and off-target toxicity. While antibody-drug conjugates (ADCs) provide a premier platform for precision oncology, achieving a high drug-to-antibody ratio (DAR) without compromising stability remains a formidable challenge for hypoimmunogenic breast cancer. Herein, we introduced a high-payload CD73 antibody-polymer-MSA-2 conjugate (αCD73-NPLG-PTM2) to potentiate breast cancer immunotherapy. By leveraging the dense carboxyl clusters of a poly(L-glutamic acid) (PLG) backbone coupled with a ROS-responsive thioketal (TK) linker for tumor-selective MSA-2 release, the average payload-to-antibody ratio was estimated to be approximately 156 by AF4-MALS/DLC analysis. Intravenous αCD73-NPLG-PTM2 is specifically recognized and internalized by CD73-overexpressing breast cancer cells, resulting in 2.27-fold higher tumor accumulation than the process-matched non-targeting isotype control. This targeted delivery simultaneously blocks CD73-mediated immunosuppression and activates STING signaling, thereby driving type I IFN-mediated antitumor responses and durable immunological memory. In murine subcutaneous E0771 and orthotopic triple-negative 4T1 tumor models, αCD73-NPLG-PTM2 effectively remodeled the immunosuppressive microenvironment and primed T cell-mediated antitumor immunity, thereby achieving potent tumor regression and an 89.6% reduction in lung metastases. This study presents a novel preparation strategy of antibody-polymeric-STING agonist conjugate for breast cancer immunotherapy. • Developed a high-payload antibody-polymer conjugate (αCD73-NPLG-PTM2) with an average payload-to-antibody ratio estimated at approximately 156 by AF4-MALS/DLC. • Synchronized CD73 blockade and STING activation effectively neutralize adenosine-mediated immunosuppression to enhance innate immune responses. • Targeted delivery via CD73-mediated internalization resulted in 2.27-fold higher tumor accumulation compared with the process-matched non-targeting isotype control. • A ROS-responsive thioketal linker ensures tumor-selective release of the STING agonist MSA-2, minimizing potential systemic toxicity. • Treatment remodeled the tumor microenvironment by promoting dendritic cell maturation, increasing M1 macrophage polarization, and depleting regulatory T cells. • αCD73-NPLG-PTM2 administration achieved potent tumor regression in E0771 and 4T1 models and reduced pulmonary metastasis by 89.6%.