An engineered pneumolysin ΔA146 derivative induces apoptosis in breast cancer cells: an in vitro proof-of-concept for antibody-drug conjugate payload development

Abstract

The therapeutic potential of antibody-drug conjugates (ADCs) is frequently undermined by the emergence of resistance to conventional payloads, which predominantly exert their effects through the disruption of intracellular targets such as microtubules or nuclear DNA. In pursuit of therapeutic agents capable of bypassing these established resistance pathways, we turned our attention to the engineered bacterial pore-forming toxin, pneumolysin ΔA146 (PLY ΔA146). To evaluate its intrinsic cytotoxic potential independent of antibody-mediated delivery, we generated a recombinant fusion protein (CPP-PLY ΔA146) for direct cellular assessment. This construct demonstrated proficient cellular internalization into both HER2-positive SK-BR-3 and triple-negative MDA-MB-231 breast cancer cell lines. Subsequent functional analysis revealed a potent and concentration-dependent suppression of cell proliferation, yielding half-maximal inhibitory concentrations (IC₅₀) of approximately 1.140 µM and 1.433 µM, respectively, after a 72-hour exposure. Mechanistically, the observed cytotoxicity was fundamentally linked to the induction of programmed cell death. This was substantiated by a convergence of evidence: a significant elevation in Annexin V-positive populations, conclusive detection of DNA strand breaks, the cleavage and consequent activation of caspase-8 and caspase-3 along with a corresponding increase in their catalytic activities, a decisive shift in the Bax/Bcl-2 equilibrium toward a pro-apoptotic phenotype. Collectively, these results delineate a defined and efficacious pro-apoptotic mechanism for PLY ΔA146, one that is functionally discrete from the modalities of current ADC mainstays. These results provide an in vitro proof-of-concept for PLY ΔA146 as a potential candidate payload for future ADC development, proposing a strategic avenue to surmount existing limitations and potentially augment the efficacy of targeted oncological interventions.