Abstract 3424: DuoBody®-FAPαxDR4 induces tumor cell death through FAPα-dependent, DR4 transactivation-mediated apoptosis

Abstract Apoptosis can be induced through activation of death receptors (DRs), which, upon binding to their natural ligands, hypercluster to initiate caspase-dependent cell death. This process can be mimicked by agonistic antibodies, making it a promising approach for anticancer therapy. However, DR-targeting cancer therapies evaluated in the clinic have shown limited success, due to insufficient clinical activity and/or safety concerns primarily related to hepatotoxicity. We hypothesize that conditional activation of DR4 in the tumor microenvironment (TME) can improve the therapeutic window of DR agonism. Fibroblast activation protein (FAP)α-expressing cancer-associated fibroblasts (CAFs) are abundant in the stroma of many solid cancers, making FAPα an attractive target to direct DR4 agonism to the TME. Here we present the novel Fc-silenced bispecific antibody DuoBody-FAPαxDR4 (GEN1057) designed to simultaneously bind to DR4 expressed on tumor cells and FAPα expressed on CAFs, to conditionally activate DR4 on the tumor cells. Functional activity of DuoBody-FAPαxDR4 was assessed in tumor cell lines and colorectal carcinoma (CRC) patient-derived organoids (PDOs) using viability and bioluminescent reporter assays and potential hepatotoxicity was evaluated using human primary liver spheroids in vitro. In vivo, antitumor activity of DuoBody-FAPαxDR4 was evaluated in patient-derived xenograft (PDX) models from different tumor types and in a model for spontaneous multiorgan metastasis of CRC. DuoBody-FAPαxDR4 induced dose-dependent caspase-8 activation and cytotoxicity in DR4-expressing tumor cell lines and CRC PDOs, when cocultured with FAPα-expressing cells in vitro. Cytotoxicity was strictly dependent on the presence of FAPα-expressing cells, with no direct effect on their viability. Furthermore, no cytotoxicity was observed in cocultures treated with monospecific control antibodies containing either the FAPα- or DR4-specific Fab arm of DuoBody-FAPαxDR4, confirming that binding to both DR4 and FAPα is required for DR4 transactivation. DuoBody-FAPαxDR4 induced caspase-8 activation and cytotoxicity in the cocultures to levels in the same range as an antibody that induced unconditional DR4 hyperclustering. Importantly, DuoBody-FAPαxDR4 did not induce cytotoxicity in human primary liver spheroids. In vivo, DuoBody-FAPαxDR4 showed potent antitumor activity in PDX models and against multiorgan metastases in a CRC PDO cecum transplantation mouse model. In conclusion, DuoBody-FAPαxDR4 is a novel bispecific antibody that activates DR4 on cancer cells exclusively in the presence of FAPα-expressing cells, leading to targeted caspase activation and tumor cell death. DuoBody-FAPαxDR4 represents a promising therapeutic agent for the treatment of cancer and is currently under evaluation in a first-in-human trial in patients with solid tumors (NCT06573294). Citation Format: Ilse Jongerius, Jamila Laoukili, Bart de Goeij, Edouard Souteyrand, Grietje Andringa, Madelon Paauwe, Monique Luijten, David Satijn, Esther Breij, Onno Kranenburg, Marije Overdijk. DuoBody®-FAPαxDR4 induces tumor cell death through FAPα-dependent, DR4 transactivation-mediated apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3424.