重编程非致病性大肠杆菌表面结构以创建用于强效癌症治疗的新一代免疫接合器

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.