LIME: A first-in-class of live bacterial immune engagers for cancer immunotherapy Free

Background: While current immune cell engagers have shown therapeutic promise, systemic exposure can lead to cytokine release syndrome (CRS) and on-target off-tumor effects. Their short half-life and the immunosuppressive tumor microenvironment (TME) limit tumor immune infiltration and therapeutic efficacy. Microbial-based cancer therapy utilizing tumor-homing bacteria offers an alternative delivery strategy to overcome challenges such as immune suppression within the TME and limited tumor infiltration.

Building on our recently developed surface display platform using non-pathogenic E. coli K-12 DH5α for cytokine delivery within the TME (Yang et al., Nat Biotech, 2024), we introduce a new class of Live Immune Modulating Engagers (LIME) that display single-chain variable fragments (scFvs) targeting both tumor-associated antigens (TAAs) and immune cell (T or NK) receptors. We hypothesize that dual surface display of anti-tumor and anti-immune effector scFvs on tumor-homing E. coli will enhance anti-tumor immunity by promoting immune activation and targeted engagement.

Methods: We engineered E. coli to express scFvs targeting TAAs (e.g., CD19, CD123, Mesothelin (MSLN), HER2, and EGFR) and immune receptors (e.g., CD3, NKG2D, CD16), fused to various scaffold proteins to optimize surface display, confirmed by flow cytometry. In vitro, engineered E. coli were co-cultured with primary human T or NK cells and tumor cells to assess bacterial binding, immune activation (CD25, CD69, Ki67), and cytotoxicity (7-AAD/Annexin V) by flow cytometry. For in vivo studies, syngeneic mouse models bearing subcutaneous tumors, KPC for PDAC MSLN⁺, A20 for B cell lymphoma CD19⁺, were treated with intravenous (i.v.) injections of PBS, non-LIME E. coli, engineered LIME E. coli (1-2×10⁸ Colony-Forming Unit (CFU)), intraperitoneal (i.p.) anti PD-1 (8 mg/kg, 2x weekly) and LIME plus anti PD-1. Tumor growth was monitored bi-weekly via caliper measurements. Tumors and organs (liver, spleen, lymph nodes) were harvested for flow cytometry, confocal imaging, multiplex ELISA, and CFU quantification.

Results: We successfully engineered E. coli to surface-display single and dual scFv constructs using various scaffold proteins, with Intimin Eaea (Neae) demonstrating the highest binding affinity and strongest activation of T and NK cells. Two distinct LIME construct groups were engineered: T-LIME, displaying an anti-CD3 scFv for T cell engagement, and N-LIME, displaying an anti-NKG2D scFv for NK cell activation. Both platforms were able to co-display scFvs targeting TAAs. These dual-display LIME robustly activated (CD25 and CD69 positivity) primary human T and NK cells and significantly enhanced their cytotoxicity (7AAD/AnnexinV positivity) against multiple tumor cell lines. Anti-CD19 and anti-MSLN N-/T-LIME were used for examining in vivo efficacy with or without anti PD-1 blockade treatment. For immunocompetent mice bearing A20 lymphoma, two i.v. injections of T- or N-LIME along with anti PD-1 i.p. was safe, led to dramatic tumor shrinkage (p<0.0001) with 80-100% mice surviving long term (>40 days to date) vs no control in scaffold only E. coli, minimal control in anti-PD1 cohort, and modest control (40% of mice cured) in the LIME cohort. We observed similar results in terms of enhanced tumor control with LIME plus anti PD-1 blockade in the KPC model, though none of these mice were cured. The engineered E. coli enriched up to 10¹² CFU/g in the tumor, 10⁷-fold more compared to spleen, liver, and kidney. Multiplex ELISA analysis showed that LIME triggered a strong immune response within the TME and with minimal increase in CRS indicating cytokines in the peripheral blood. Confocal imaging of the tumor tissue showed increased infiltration of T and NK cells in the mice treated with the LIME and confirmed enrichment of engineered E. coli in the necrotic regions of the tumor and infiltration of the engineered E. coli in non-necrotic tumor tissue.Conclusions: Here we describe the development of LIME, a novel platform for targeted cancer immunotherapy, using non-pathogenic E. coli to display bispecific scFvs that activate T and NK cells and drive tumor-specific killing with minimal systemic toxicity. In vivo, LIME synergize with anti PD-1 to induce robust and durable tumor control, highlighting their potential as a safe, modular, and tumor-selective immunotherapy platform.