Augmented antitumor immune responses of HER2-targeted pyroptotic induction by long-lasting recombinant...

Pyroptosis is a well-documented form of programmed cell death caused by the gasdermin-driven perforation of cell membranes. Selective induction of pyroptosis in tumor cells represents a promising antitumor strategy to enhance the efficacy of immunotherapy. In this study, we established a recombinant protein-based immunopyroptotin strategy that led to the intratumoral induction of pyroptosis for HER2-directed therapy. Long-lasting immunopyroptotins were constructed by sequentially fusing the humanized anti-HER2 single-chain antibody P1h3, albumin-binding peptide (ABD035 or dAb7h8), cathepsin B-cleavable peptide B2, endosome-disruptive peptide E5C3, and active pyroptotic effector gasdermin D-N fragment (GN). After purification, we evaluated the cytotoxicity and antitumor immune responses primarily induced by the immunopyroptotins in HER2-overexpressing breast cancer cells. The resulting ABD035-immunoGN and dAb7h8-immunoGN showed improved in vitro cytotoxicity in HER2-overexpressing cancer cells compared with that in the immunotBid that we previously generated to induce tumor cell apoptosis. The binding of long-lasting immunopyroptotins to albumin increased the half-life by approximately 7-fold in nude mice. The enhanced antitumor efficacy of long-lasting immunopyroptotins was confirmed in both N87 tumor-bearing T cell-deficient mice and 4T1-hHER2 bilateral tumor-bearing immunocompetent mice. Immunopyroptotin treatment elicited systemic antitumor immune responses involving CD8⁺ T cells and mature dendritic cells and upregulated the expression of proinflammatory cytokines, leading to sustained remission of non-injected distant tumors. This study extends the repertoire of antibody-based therapeutics through the tumor-targeted delivery of a constitutively active pore-forming gasdermin-N fragment, which shows great potential for pyroptosis-based antitumor therapy.