In vitro and in vivo evaluation of the bystander-killing effect of ADCs

Antibody-drug conjugates (ADCs) represent a promising class of therapeutics with a broader therapeutic window than conventional chemotherapeutic agents, owing to their efficient and specific drug delivery. In addition, some ADCs produce a bystander-killing effect through mechanisms such as soluble toxic mediators, gap junction intercellular communication, immune-mediated killing, or Fc-mediated effector functions. This effect benefits not only antigen-expressing tumor cells, but also adjacent antigen-negative cells. Trophoblast cell-surface antigen 2 (Trop-2) is a transmembrane glycoprotein highly expressed in many epithelial cancers and serves as an attractive target for ADC development. In this study, we assessed the bystander-killing effect of two Trop-2-targeted ADCs-sacituzumab govitecan (SG) and datopotamab deruxtecan (Dato-DXd). To evaluate this effect, we used both in vitro co-culture assays and in vivo mixed-cell xenograft tumor models. Three cell line pairs (BxPC-3/UM-UC-3, HCC1806/UM-UC-3, NCI-N87/UM-UC-3) were selected for the in vitro assay based on Trop-2 expression. Dato-DXd exhibited higher payload release in both the BxPC-3 group and the BxPC-3/UM-UC-3 co-culture group compared to UM-UC-3 alone, which showed minimal payload release. In contrast, SG demonstrated similar payload release across all groups. Cell proliferation assays revealed that Dato-DXd had potent cytotoxicity against Trop-2-positive cells (BxPC-3, HCC1806, NCI-N87) but was ineffective in UM-UC-3 cells. Conversely, SG exhibited comparable cytotoxicity across all four cell lines. Notably, Dato-DXd reduced the viability of UM-UC-3 cells in all co-culture groups, with the BxPC-3/UM-UC-3 group showing the strongest bystander effect (80% inhibition of UM-UC-3 cells). Unlike Dato-DXd, SG exhibited strong cytotoxicity in both co-culture and UM-UC-3 groups. A similar observation was made in vivo using a mixture of BxPC-3 and UM-UC-3-Luc cell line-derived xenograft model, with tumor volume monitored via imaging. These results demonstrate that co-culture systems and in vivo imaging are valuable tools for ADC drug discovery. Both in vitro and in vivo assays are essential for understanding bystander-killing mechanisms and predicting clinical success.