Fully human anti-B7-H4 antibody induces lysosome-dependent ferroptosis to reverse primary resistance to PD-1 blockade

Background Although immune checkpoint inhibitors (ICIs) have significantly improved outcomes for patients with certain cancers, their efficacy is largely confined to “hot” tumors characterized by robust infiltration of tumor-specific CD8 + T cells. Conversely, tumors expressing B7-H4 often exhibit an immunologically “cold” tumor microenvironment with poor T cell infiltration, contributing to primary resistance to programmed cell death protein 1 (PD-1) blockade. Methods We evaluated the association between B7-H4 expression and clinical outcomes in ICI-treated patients using public immunotherapy datasets. The role of B7-H4 in mediating resistance to PD-1 therapy was examined in mouse tumor models. A fully human anti-B7-H4 monoclonal antibody (clone A8) was generated via phage display screening from a non-immunized human single-chain variable fragment library. In vitro assays assessed antibody-induced tumor cell death and immune activation, while in vivo efficacy was tested in MC38-mH4 and SKOV3-hH4 tumor models, as well as human colorectal cancer organoids. Statistical analyses included Student’s t-test, one-way analysis of variance, and Kaplan-Meier survival analysis, with p<0.05 considered significant. Results High B7-H4 expression was associated with inferior prognosis in patients receiving ICI therapy. In MC38-mH4 tumors, B7-H4 expression conferred resistance to anti-PD-1 treatment. We identified A8, a novel antibody targeting the IgV-like domain of B7-H4, with cross-reactivity to both human and mouse B7-H4. A8-hIgG1 and its Fab fragment induced dynamin-dependent endocytosis of B7-H4, resulting in lysosomal accumulation, altered lysosomal membrane permeabilization and intracellular acidification, ultimately triggering ferroptosis, a form of immunogenic cell death. A8 binding was enhanced under acidic conditions (pH 5.5), promoting lysosome-dependent degradation of B7-H4. A8-induced ferroptosis enhanced dendritic cell maturation, macrophage phagocytosis, and T cell activation. In vivo, A8 promoted CD8 + T cell and HER2 chimeric antigen receptor-T cell infiltration, inhibited tumor growth, and synergized with PD-1 blockade to overcome primary resistance in multiple preclinical models. This immunogenic and lysosome-dependent cell death mechanism was unique to A8 among the anti-B7-H4 antibodies tested. Conclusions Our study identifies a novel mechanism by which a fully human anti-B7-H4 antibody induces lysosome-dependent immunogenic tumor cell death. These findings support the therapeutic potential of A8 as a single agent or in combination with PD-1 blockade to overcome immune resistance in B7-H4-expressing “cold” tumors.