Abstract 4904: Reprogramming of the non-pathogenic <i>E. coli</i> surface architecture to create next-generation immune engagers for potent cancer therapies

Abstract Background: Multi-specific immune engagers show therapeutic promise but are limited by systemic toxicities (e.g., cytokine release syndrome, CRS), short half-lives, and poor infiltration into the immunosuppressive tumor microenvironment (TME). Tumor-homing bacteria offer a targeted delivery strategy capable of overcoming restricted infiltration and TME-mediated suppression. Building on our E. coli K-12 surface display platform for localized cytokine delivery (Yang et al., Nat Biotechnol, 2024), we developed Live Immune Modulating Engagers (LIME), engineered E. coli co-displaying scFvs against tumor-associated antigens and effector-cell receptors (T or NK). We hypothesize that dual-display LIME enhances anti-tumor immunity by enabling localized effector-cell activation and precise tumor-immune cell bridging. Methods: E. coli was engineered to display scFvs targeting TAAs (CD19, Mesothelin, DLL3, etc.) and immune receptors (CD3, NKG2D, etc.) using optimized outer-membrane scaffolds, confirmed by flow cytometry. In vitro co-cultures with primary human T or NK cells assessed binding, activation (CD25/CD69), and cytotoxicity (7-AAD/Annexin V). In syngeneic A20 and KPC tumor models, mice received intravenous LIME ± anti-PD-1 (i.p.) or RMC-7977 (oral). In DLL3+ H69 xenografts, NSG mice were infused with human CD3+ T cells followed by LIME, benchmarked against the FDA-approved engager Tarlatamab. Tumor growth was monitored and analyzed by flow cytometry, multiplex ELISA, and CFU quantification. Results: In vitro, LIME activated T or NK cells and enhanced killing across a broad panel of tumor cell lines, including Small cell lung cancer, Glioblastoma, Pancreatic ductal adenocarcinoma, etc. In vivo, CD19 LIME cured ∼34% of A20-bearing mice and achieved ∼80% cures when combined with anti-PD-1, generating durable immune memory. Mesothelin LIME significantly inhibited KPC tumor progression, with extended survival upon RMC-7977 co-treatment. DLL3 LIME provided superior tumor control compared with Tarlatamab in H69 xenografts. Mechanistically, LIME reprogrammed the TME by activating T and NK cells, driving macrophage repolarization toward an M1 state, and enhancing DC activation in draining lymph nodes. Bacteria remained enriched in tumors, and no evidence of CRS was observed in plasma. Conclusions: LIME represents a versatile platform that enables localized, immune engagement, overcomes major barriers in solid tumors, and outperforms existing immune engagers while maintaining a favorable safety profile. Citation Format: Shaobo Yang, Anna Clara Bader, Stephanie Sendker, Ashley Hu, Alice Chen, Daniel Chen, Hetal Nath, Eden Bobilev, Michal Sheffer, Veronica W. Hui, Tereza Kochs, Andreia Maia, Fuguo Liu, Xingyu Deng, Maily Nguyen, Mila Stanojevic, Mubin Tarannum, Alaa Ali, Roman Shapiro, Young Rock Chung, Erin M. Parry, Julissa G. Tello, Stephanie K. Dougan, Marco Campisi, David A. Barbie, John Koreth, Robert J. Soiffer, Catherine J. Wu, Jerome Ritz, Rizwan Romee. Reprogramming of the non-pathogenic E. coli surface architecture to create next-generation immune engagers for potent cancer therapies [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 4904.