Enhancing the bystander effect of antibody-drug conjugate by using a novel caspase-3 cleavable peptide linker to overcome tumor heterogeneity.

Tumor heterogeneity is a major obstacle to effective targeted therapies, including those utilizing antibody-drug conjugates (ADCs). Although some ADCs employ the bystander effect to eliminate neighboring antigen-negative cells, their efficacy often diminishes as antigen-positive cell populations decrease within heterogeneous tumors. To address this limitation in ADC therapies, we developed a novel ADC using a caspase substrate, Asp-Glu-Val-Asp (DEVD), as a linker to generate a more potent and sustained bystander effect. The DEVD ADC effectively targeted antigen-positive cells and released its payload via cathepsin B cleavage. Notably, it exhibited a significant bystander effect mediated by the caspase-3-triggered extracellular cleavage of the linker, enhancing payload release into the tumor microenvironment. In breast cancer xenograft models, the DEVD ADC maintained its efficacy and continued to exert a bystander effect even after the depletion of antigen-positive cells, thereby overcoming challenges posed by tumor heterogeneity. These findings emphasize the potential of DEVD linkers in enhancing ADC efficacy against heterogeneous solid tumors, offering a promising strategy to improve therapeutic outcomes.