Abstract B085: Lysosome-dependent ferroptosis induced by a fully human anti-B7-H4 antibody overcomes primary resistance to PD-1 blockade

Abstract Purpose Although immune checkpoint inhibitors (anti-PD-L1, anti-PD-1 and anti-CTLA-4) have demonstrated to extend life expectancy in patients with certain cancers, their efficacy is limited to tumors with a substantial infiltration of tumor-specific T cells, a phenomenon known as "hot" tumor. Our previous study and others demonstrated that B7-H4 expression in tumor cells limits CD8 T cell infiltration, creating an immunosuppressive "cold" tumor microenvironment. Hence, we sought to generate an immunomodulatory antibody to target cell surface B7-H4 and investigate its anti-tumor mechanism in "cold" tumors. Methods B7-H4-mediated primary resistance to anti-PD-1 antibody was investigated in B7-H4 overexpressing mouse tumor (MC38-mH4) models and transcriptomic datasets of cancer patients treated with immune-checkpoint inhibitors. Next, we developed an immunomodulatory anti-B7-H4 antibody using a non-immunized human scFv library. The binding domains of B7-H4 and cross-reactivity were examined by B7-H4-expressing tumor cells. The direct effect of anti-B7-H4 antibodies on tumor cell lines was evaluated. Furthermore, the blockade activity of the anti-B7-H4 antibody on T cell proliferation and cytotoxicity was assessed in vitro. The antitumor efficacy of the anti-B7-H4 antibody and its synergistic effect with PD-1 antibodies was evaluated in MC38-mH4 models, SKOV3-hH4 models and human colorectal cancer organoids. Results Based on Kaplan-Meier Plotter, we found high B7-H4 expression was related to unfavorable prognosis of patients receiving immunotherapy. B7-H4-mediated primary resistance to anti-PD-1 antibody was confirmed in MC38-mH4 models. Subsequently. we screened the A8 clone, which targets the Ig-like V-type domain of B7-H4 and exhibits cross-binding ability to both human and mouse B7-H4. Notably, both a full-length human IgG1 antibody (A8-hIgG1) and Fab fragment (A8-Fab) induced dynamin-dependent endocytosis and lysosomal degradation of B7-H4, leading to ferroptosis in several B7-H4 positive tumor cell lines. A8-induced immunogenic cell death promoted dendritic cell maturation and T cell activation. Additionally, A8-hIgG1 enhanced T cell cytotoxicity via neutralizing effect but not ADCC effect of NK cells. More importantly, A8 facilitated the infiltration of CD8+ T cells or HER2 CAR-T cells into the tumor sites, inhibiting tumor growth in MC38-mH4 models, SKOV3-hH4 models and human colorectal cancer organoids, while overcoming B7-H4-mediated primary resistance to anti-PD-1 antibody. Importantly, this distinctive mechanism was exclusively observed with A8 compared to other anti-B7-H4 antibodies tested. Conclusions These data provide evidence for the potential utility of A8 for treatment of B7-H4-expressing "cold" tumor and support the rationale for the combination of A8 with PD-L1/PD-1 antibodies therapy. In addition, our study revealed a unique immunogenic effect for anti-B7-H4 antibody, provides a new perspective for the development of therapeutic antibodies and the combination immunotherapy. Citation Format: Renlu Zhang, Yang Wang, Yue Wu, Bingyu Zheng, Qiumei Cao, Yunhan Chen, Yanyang Zhu, Linlin Zhou, Qiuyu Zhang. Lysosome-dependent ferroptosis induced by a fully human anti-B7-H4 antibody overcomes primary resistance to PD-1 blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B085.