Abstract 480: An engineered bacterial delivery platform, BacID, selectively delivers activated Caspase-3 into the cytoplasm of cancer cells, eliminating tumors and reducing metastatic burden

Abstract Most cytotoxic therapies, either administered as a compound or incorporated as cargo into a targeted platform (nanoparticles, antibody drug conjugates etc.), only become effective after reaching the intended target inside the tumor cell cytoplasm. Intracellular targeting faces multiple challenges, including tumor-specific receptor-binding, crossing the cell membrane, and endosomal escape into the cytoplasm. Each of these limitations reduce tumor accumulation while increasing systemic toxicity of the drug. Here, we have created a clinically-ready, non-pathogenic strain of bacteria, BacID, that accumulates and grows exponentially in tumors. The bacteria have a 5, 000-fold selectivity for tumors over healthy organs and enter into cancer cells without requiring any tumor-specific receptor engagement. Once intracellular, BacID lyse and deliver a cytotoxic payload into the cytoplasm. The resulting cell death creates an immunostimulatory microenvironment that facilitates tumor clearance. The BacID strain is engineered to be safe by attenuating the external lipopolysaccharide layer, flagella expression and utilizing the intracellular lysis system that enables both delivery inside cancer cells and autonomous clearance after treatment. The bacteria is well tolerated after systemic administration in mice. To target liver cancer and metastases, The BacID strain was engineered to deliver activated Caspase-3 (EBT-002) to cancer cells. After systemic administration, the bacteria colonize tumors and rapidly clear from healthy organs. Three days after injection, bacterial concentrations in tumors peak. At this point, aspirin is administered to activate expression of flagella and the type 3 secretion system, which triggers the BacID strain to invade cancer cells. After invasion, the bacteria enter a modified endosome within the cell, lyse, and deliver the activated Caspase-3 payload into the cytoplasm, inducing cell death. Delivery of Caspase-3 with BacID enables tumor clearance, in part, by activating a 12-fold increase in intratumoral innate immunity, followed by an adaptive immune response. EBT-002 also targets and eliminates liver and lung metastases of breast cancer as small as 100 µm in diameter. After systemic administration, EBT-002 reduces lung metastatic burden by over 80% compared to a standard-of-care chemotherapeutic regimen. The BacID platform enables delivery of several biological therapies selectively inside tumor cells due to the natural tumor tropism of the platform. Moreover, the BacID strain can express and deliver DNA, RNA and protein-based therapies into tumor cells without any need for tumor-specific receptor engagement due to the natural cell invasion mechanisms inherent to the platform. These qualities make the BacID platform a universal delivery vehicle to treat solid tumors. Citation Format: Vishnu Raman, Christopher L. Hall, Victoria E. Wetherby, Neil S. Forbes, Nele Van Dessel. An engineered bacterial delivery platform, BacID, selectively delivers activated Caspase-3 into the cytoplasm of cancer cells, eliminating tumors and reducing metastatic burden [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 480.