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 trophoblast cell surface antigen 2 (TROP2) in PDAC, we developed a TROP2-targeted Toll-like receptor 7 (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, nonglycosylated immune-stimulating antibody conjugates (NG-ISAC) induce robust myeloid activation, cytokine release, and tumor regression without FcγR-mediated functions of NK 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 cocultures of TROP2-positive tumor and effector cells. Moreover, NG-anti-TROP2-E104-ISACs display reduced acute toxicity compared with glycosylated counterparts. Together, these findings delineate the bystander mechanism underlying FcγR-independent immune stimulation and establish a framework for designing ISACs with improved safety.