ZHPV16E7-GrB Affitoxin Induces Caspase-3-Independent GSDME-Mediated Pyroptosis, Suppressing HPV16-Positive Tumors

Persistent infection with high-risk human papillomavirus (HPV), particularly HPV16, is the primary driver of cervical cancer progression. The viral E7 oncoprotein is continuously overexpressed in HPV16-positive cells, making it an attractive and specific therapeutic target. Current treatments often lack the specificity to target HPV-infected cells while sparing healthy tissue, leading to off-target effects. There's a critical need for novel, targeted therapies that can selectively eliminate HPV16-positive cancer cells by exploiting unique viral protein expression.

Researchers developed a novel affitoxin, ZHPV16E7-GrB, by fusing a cell-penetrating peptide (CPP) to an affibody (ZHPV16E7) specific for the HPV16E7 protein, then conjugating it with human granzyme B (GrB). This fusion protein was successfully prepared in a prokaryotic system. Its specific binding to the HPV16E7 protein and rapid, highly specific cellular uptake in HPV16-positive cervical cancer cells were confirmed. The therapeutic efficacy was evaluated in HPV16-positive TC-1 xenograft mouse models in vivo, observing effects on tumor growth and pyroptosis induction.

ZHPV16E7-GrB demonstrated specific binding to HPV16E7 and rapid, highly specific cellular uptake in HPV16-positive cervical cancer cells. Significantly, ZHPV16E7-GrB induced obvious pyroptosis, characterized by distinct cell membrane bubble-like protrusions, gasdermin E (GSDME) cleavage, and the release of proinflammatory cytokines such as IL-18, IL-1β, and HMGB1, alongside LDH release. Importantly, this pyroptosis occurred independently of caspase-3 activation, a finding confirmed by caspase-3 knockdown experiments, highlighting GSDME as the key executor. > Experimental studies in vivo showed that pyroptosis was likely involved in the significant suppression of tumor growth in HPV16-positive TC-1 xenograft mouse models treated with ZHPV16E7-GrB.