Abstract 5794: Tumor cell targeted granzyme B fusion constructs mediate immunogenic cell death (ICD) in addition to their direct cytotoxic effects

Abstract Therapeutic agents targeting tumor cells have a cytotoxic effect that primarily results in an apoptotic insult through numerous mechanisms. Recently, immune activation mechanisms directed against tumor cells have been identified as a secondary effect of targeted therapy. In this mechanism, targeted tumor cells release ATP and various intracellular proteins into the intratumoral space including HSP70, HMGB1 and calreticulin. These damage-associated molecular patterns (DAMPs) then decorate nearby undamaged tumor cells where infiltrating host immune cells generate a secondary immune response to these decorated bystander cells. Epithelial membrane protein-2 (EMP2) is highly expressed in invasive breast cancer (BC), including triple negative breast cancer, and represents an attractive therapeutic target. We generated a fusion construct containing human granzyme B fused to an scFv antibody recognizing the EMP2 antigen, tethered through a fragment of an IgG heavy chain (Fc) domain and containing the dimerization domain. The 160 kDa dimeric protein was expressed by HEK-293E cells into conditioned media. The construct was assessed for GrB enzymatic activity, EMP2 binding affinity and cytotoxicity against various breast cancer cell lines. The purified GrB-Fc-KS49 demonstrated exceptional binding to an EMP2 peptide with a KD of 1 x 10-12 M and GrB enzymatic activity was preserved compared to native GrB. The IC50 ranged from 40-83 nM against a panel of murine and human EMP2+ BC cell lines. Treatment of mice bearing well-established syngeneic EMT6 or 4T1 breast tumors on a twice weekly schedule (IP, 40 mg/kg total dose) showed a significant (50%) reduction in cell growth and in tumor cell turnover (Ki-67). Importantly, in the 4T1 model tumors, there was a 20-fold increase in CD45+ immune cells and a 60-fold increase in tumor-inhibitory M1 macrophages. We found similar results in the EMT6 tumor model with a significant decrease in tumor supportive M2 macrophages. Studies are ongoing to identify potential directly cytotoxic molecules released from cells intoxicated by GrB-based fusion constructs and to identify 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 part, by the Clayton Foundation for Research and generously supported by the NCI R21 CA163971. Citation Format: Khalid A. Mohamedali, Brian Aguirre, Lawrence H. Cheung, SuYin Kok, David Casero, Madhuri Wadehra, Michael G. Rosenblum. Tumor cell targeted granzyme B fusion constructs mediate immunogenic cell death (ICD) in addition to their direct cytotoxic effects [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 5794.