A pneumolysin mutant (ΔA146 ply) induces pyroptosis in triple-negative and HER2-positive breast cancer cells via the Caspase-1/GSDME pathway: a potential antibody-drug conjugate payload.

Background: The success of antibody-drug conjugates (ADCs) relies on potent cytotoxic payloads. Discovery of novel toxins that induce immunogenic cell death (such as pyroptosis) holds promise for overcoming tumor heterogeneity and enhancing anti-tumor immunity. Pneumolysin (Ply) is a pore-forming toxin from Streptococcus pneumoniae, but its wild-type form is overly toxic. Here we investigate the potential of a detoxified mutant, ΔA146 ply, to induce pyroptosis in triple‑negative (MDA‑MB‑231) and human epidermal growth factor receptor 2 (HER2)‑positive (MDA‑MB‑453) breast cancer cells, using a cell-penetrating peptide (CPP) fusion to facilitate cellular entry for in vitro evaluation of its activity as a candidate ADC payload. Methods: A flexible peptide linker was used to fuse ΔA146 ply with a CPP to facilitate cellular uptake, and the fusion gene was cloned into the pET‑21a(+) prokaryotic expression vector. The recombinant fusion protein was expressed in E. coli and purified by affinity chromatography. Cellular internalization was assessed by immunofluorescence using FITC-labeled protein. Cell death modalities were evaluated by lactate dehydrogenase (LDH) release assay, SYTOX Green staining, and morphological observation. Western blotting was performed to detect the cleavage of the pyroptosis executioner gasdermin E (GSDME). Caspase-1 activity was measured in cell lysates, and the release of inflammatory factors [interleukin‑1β (IL‑1β), interleukin‑18 (IL‑18), and high mobility group box 1 (HMGB1)] in the cell supernatant was determined. Results: High-purity recombinant ΔA146 ply-CPP was successfully obtained. The protein efficiently entered MDA-MB-231 and MDA-MB-453 cells. ΔA146 ply-treated cells displayed typical pyroptotic morphology (cell swelling, large bubble-like protrusions), significantly increased LDH release, and positive SYTOX Green staining. Mechanistically, ΔA146 ply markedly activated Caspase-1, induced GSDME cleavage, and promoted the release of IL-1β, IL-18, and HMGB1. Similar effects were observed in both cell lines, with no qualitative difference in the pyroptotic mechanism. Conclusions: ΔA146 ply triggers pyroptosis in breast cancer cells through the Caspase-1-mediated GSDME pathway. As a payload with immunostimulatory potential, it warrants further development for ADC applications.