Nonglycosylated, Legumain-Cleavable ISACs Drive Potent Antitumor Immunotherapy via a Bystander Effect

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal form of pancreatic cancer, with poor prognosis driven by late diagnosis, therapeutic resistance, and an immunosuppressive tumor microenvironment. Interactions between tumor cells and immune cells promote immune evasion and tumor progression, limiting the efficacy of immune checkpoint blockade and other immunotherapies. Given the high expression of TROP2 in PDAC, we developed a TROP2-targeted TLR7 agonist (E104) designed for selective accumulation within the tumor microenvironment to activate antitumor immunity. Although antibody bioactivity is traditionally linked to Fcγ receptor (FcγR) engagement and recruitment of effector cells, our legumain-cleavable, non-glycosylated immune-stimulating antibody conjugates (NG-ISACs) induce robust myeloid activation, cytokine release, and tumor regression without FcγR-mediated functions of natural killer cells or macrophages. Rather, NG-anti-TROP2-E104-ISACs depend on tumor antigen recognition and TLR7 activation, not FcγR-driven antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), to elicit tumor regression and adaptive immunity, as evidenced by anti-TROP2 antibody generation in a syngeneic model. By incorporating the cell-permeable E104 payload with bystander activity, NG-ISACs can activate immune responses independently of FcγR binding. In vitro, NG-anti-TROP2-E104-ISACs bypass FcγRIIa-mediated ADCC and FcγRIIIa-mediated ADCP while maintaining potency in co-cultures of TROP2-positive tumor and effector cells. Moreover, NG-anti-TROP2-E104-ISACs display reduced acute toxicity compared to glycosylated counterparts. Together, these findings delineate the bystander mechanism underlying FcγR-independent immune stimulation and establish a framework for designing ISACs with improved safety.