HPV16E7-specific affitoxin induces GSDME-mediated pyroptosis via a caspase-3-independent pathway

The human papillomavirus (HPV) E7 oncoprotein drives cervical cancer progression through sustained overexpression in HPV16-positive cells, making it an ideal therapeutic target. This study developed a novel affitoxin (Z_HPV16E7-GrB) to target HPV16E7 and elucidated its antitumor mechanism in HPV16-positive cervical cancer.

Z_HPV16E7-GrB was developed by fusing a cell-penetrating peptide (CPP) to an affibody (Z_HPV16E7) specific for HPV16E7 and conjugating it with human granzyme B (GrB), a cytotoxic effector molecule. Its binding specificity, cellular uptake, and ability to induce pyroptosis were evaluated using in vitro assays. The underlying mechanisms were investigated using siRNA-mediated knockdown and pan-caspase inhibition, and its in vivo antitumor efficacy was assessed in a TC-1 syngeneic tumor model.

The purified Z_HPV16E7-GrB exhibited specific binding to the HPV16E7 protein and rapid cellular uptake in HPV16-positive cervical cancer cells. Treatment with Z_HPV16E7-GrB clearly induced pyroptosis, characterized by bubble-like protrusions of the cell membrane, GSDME cleavage, and the release of proinflammatory cytokines (IL-18, IL-1β, and HMGB1) and LDH. Importantly, this pyroptosis occurred independently of caspase-3 activation, as demonstrated by caspase-3 knockdown and pan-caspase inhibition, with GSDME identified as the key executor of pyroptosis. Consistently, Z_HPV16E7-GrB significantly suppressed tumor growth in a TC-1 syngeneic tumor model, with pyroptosis likely contributing to its antitumor activity.

Our findings provide mechanistic insights into a previously unrecognized antitumor pathway triggered by Z_HPV16E7-GrB, which directly regulates pyroptosis mediated by GSDME cleavage by bypassing the caspase-3 activation pathway, revealing a novel approach for HPV16-specific cancer therapy.