Abstract 5404: Tumor cell targeted granzyme B demonstrates direct cytotoxic action and indirect immunogenic cell death mediated through numerous defined proteins.

Abstract The primary effect of antibody-directed therapeutics is the delivery of cytotoxic payloads directly to antigen-expressing tumor cells resulting in activating cell death mechanisms in the target cells while sparing nonmalignant cells and reducing systemic toxicity. Cytotoxic payloads with the additional capacity to initiate immune activation mechanisms directed against tumor cells as a secondary effect of targeted therapy have the added benefit of initiating “immunogenic cell death” (ICD), which allows further control over residual tumor cells which may be antigen-negative or resistant to the initial cytotoxic effect. Our previous studies in two syngeneic breast cancer mouse models showed that an EMP2-targeted granzyme B fusion significantly enhanced the tumor levels of CD45+ immune cells and tumor-inhibitory M1 macrophages, as well as decreased tumor-supporting M2 macrophages. Tumor cells undergoing ICD express and secrete immunogenic factors such as damage-associated molecular patterns (DAMPs) that are critical in immune cell recruitment and the initiation of immunogenic apoptosis. Accordingly, we investigated whether targeted granzyme B delivery into human tumor cells resulted in the release or expression of DAMP proteins. We tested three human cell lines with varying levels of antigen expression for FOLRa, Fn14, EMP2 and HER2 with scFv-based constructs targeting each of these antigens and delivering the cytotoxic payload granzyme B. In vitro cytotoxicity against each cell line for all four constructs was found to be in the low nanomolar range. Treatment with all four constructs over 72 hours against log-phase tumor cells resulted in the detection of HMGB1 in the conditioned media while detection of released Annexin A1 was restricted to HEY A8 cells targeted via FOLRa. We characterized HMGB1 and Annexin A1 release into the media in a dose dependent (0-100 nM) and time-dependent (4-72h) manner and correlated these events with real-time release of ATP into the conditioned media. Furthermore, we characterized the expression of Calreticulin on the cell surface of each of these cell lines by flow cytometry and investigated the onset of caspase activation by fluorescence imaging. Further studies are ongoing to identify other DAMP-related proteins which may mediate the secondary immunogenic bystander effect against tumor cells. These data support a proposed “dual mechanism” for GrB-based therapeutics which include a direct, antigen-mediated cytotoxic effect and a secondary, immune-mediated effect which can target both antigen-positive and antigen-negative tumors. Research conducted, in whole or in part, by the Clayton Foundation for Research and generously supported by the NCI R21 CA163971. Citation Format: Khalid A. Mohamedali, Madhuri Wadehra, Lawrence H. Cheung, Michael G. Rosenblum. Tumor cell targeted granzyme B demonstrates direct cytotoxic action and indirect immunogenic cell death mediated through numerous defined proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5404.