Abstract 3439: An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts (2022)

Abstract Carcinoembyronic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in numerous solid tumors. CEA is constitutively released from tumor cells and reaches detectable levels in peripheral blood, making CEA targeting particularly challenging. Granzyme B is a key effector in immune-mediated killing of target cells, with cell death through both caspase-dependent and -independent mechanisms. We developed a fusion protein composed of granzyme B and a human scFv targeting the juxta-membrane epitope of CEA. The construct also incorporates a fragment of human IgG heavy chain to cause dimerization and provide increased in vivo circulation and antitumor efficacy. The GrB-Fc-CEA construct was cloned into a mammalian expression vector, transiently expressed by HEK-293E suspension cells, and secreted into the culture media. The final yield after purification and activation was 10-15 mg/L. The enzymatic activity of granzyme B in GrB-Fc-CEA was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against CEA-positive cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar range cytotoxicity against control (CEA-negative) cell lines. Importantly, GrB-Fc-CEA cytotoxicity against tumor cells in vitro was unaffected by exogenously-added CEA (200 ng/ml) extracellular domain, suggesting that the construct is not competitively absorbed by soluble CEA released by tumors and present in the circulation of patients. GrB-Fc-CEA internalized rapidly (within one hour) into the target cell lines A-431 and HT-1080/CEA whereas internalization was not observed with untargeted granzyme B, clearly demonstrating receptor-mediated internalization. GrB-Fc-CEA induced mitochondrial depolarization and caspase activation, consistent with the well-described granzyme B mechanism of action. Treatment of female nude mice bearing A-549 tumors with GrB-Fc-CEA (80 mg/kg over 5 doses) resulted in a significant growth inhibition of the established tumors compared to vehicle controls (1200 mm3 vs &amp;lt;40 mm3). Complete tumor regression was observed in seven of ten tumors in mice treated with GrB-Fc-CEA. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Thus, GrB-Fc-CEA appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3439.