Dual-function engineered bacteria remodel AML tumor microenvironment via IL-12 delivery and adenosine depletion to sustain NK cell immunity

Abstract

Abstract Background Cellular immunotherapy has revolutionized cancer treatment. Yet its efficacy in acute myeloid leukemia (AML) remains limited due to an immunosuppressive tumor microenvironment (TME) often characterized by deficient immune-stimulatory cytokines and elevated immunosuppressive metabolites. Adenosine (Ado), a key inhibitor is degraded by the non-redundant enzyme adenosine deaminase (ADA), which converts Ado into the immune-stimulatory inosine (Ino). IL-12 is a potent cytokine that strongly boosts immune cells, but its systemic toxicity limits clinical use. To address this, we engineered tumor-homing, non-pathogenic Escherichia coli (E. coli) to co-display surface-tethered IL-12 and ADA, enabling localized cytokine delivery and metabolic reprogramming. We hypothesized that this dual strategy efficiently reprograms the immunosuppressive niche in AML, thereby unlocking robust local NK (and T cell) anti-leukemic immunity. Method Non-pathogenic E. coli K12 DH5α were engineered to surface-display murine and human IL-12, ADA, or IL-12+ADA fusion using optimized outer membrane scaffolds with FLAG tags using an approach in our recently published paper (Yang et al, Nature Biotechnology 2024). Expression was induced with rhamnose and confirmed by flow cytometry. IL-12 bioactivity was validated via HEK-Blue reporter assay. ADA kinetics and activity were assessed via LC-MS and colorimetric assays. NK cells were co-cultured for 24h with E. coli displaying ADA, IL-12, ADA+IL-12, or scaffold control followed by multicolor flow cytometry to assess Ado-signaling (A2AR, CD73, CD39, CD26), surface markers (i.e., CD25, NKG2D, NKp30), and AML-directed cytotoxicity (Zombie-NIR, IFNg, TNFα) against the AML targets NOMO1 and OCI-AML3. To model TME metabolism, cells were treated with 1mM Ado, Ino or PBS. In-vivo efficacy was tested in a immunocompetent B16-F10 melanoma model and BALB/c mice bearing GFP⁺/NanoLuc⁺ WEHI-3 AML cells. Leukemic burden (bioluminescence imaging, blood), weight, and survival were monitored. Bacterial distribution was assessed by CFU assays in relevant organs. Results Engineered E. coli co-displaying IL-12 and ADA showed robust FLAG-tagged surface expression (>80%) with significant IL-12 bioactivity (OD650, 0.9 vs. 0.2, p<.001). Murine ADA displayed by YiaT181 showed highest enzymatic activity (Km=0.05, Vmax=53µmol/min), fully converting Ado to Ino within 4h. NK cells cocultured with IL-12 displaying E. coli showed strong upregulation of key activation markers compared to scaffold control (CD25, 56 vs. 25% and NKp30, 68 vs. 35%, p<.001), leading to a ≥3.4-fold greater AML-directed cytotoxicity and IFNγ production. Importantly, rhIL-12 (50ng/ml) increased expression of the Ado-generating metabolic checkpoint CD73 (2 to 9%, p=.003) and Ado receptor A2AR on NK cells (31 to 51%, p=.001), indicating increased Ado susceptibility. Ado exposure reduced AML-directed killing by ~50% (p<.001) and NK cell activation (NKp30 expression was 33 vs. 18%, p=.003). ADA-expressing E. coli restored NK cell immunity and AML lysis in-vitro (55 vs. 15%, p<.001). Co-display of IL-12 and murine ADA maintained full NK effector function under Ado-rich conditions and enhanced AML-targeted lysis (15 vs. 56%, p<0.01). In-vivo, i.v. delivery of E. coli expressing ADA, IL-12, or ADA+IL12 fusion was well-tolerated and demonstrated potent antileukemic activity in immunocompetent WEHI-3 AML models. Substantial tumor control led to prolonged survival in the IL-12+ADA group (2 of 3 alive at d29, median survival not reached) vs a median of 10 days in control mice. Robust tumor control was reached in the IL-12 (26d) and ADA (17d) group. Post-mortem analysis of mice revealed unprecedented tumor-guided bacterial migration to the bone marrow, a phenomenon not previously reported. ADA-engineered bacteria accumulated up to 4x105 CFU and IL-12 displaying bacteria up to 3x104 CFU in the bone marrow. Intratumoral delivery of IL12+ADA-engineered E. coli induced complete tumor regression and 100% survival in a B16-F10 melanoma model, with tumor rechallenge confirming durable antitumor immunity, suggesting immune memory and long-term immune protection. Conclusion Engineered nonpathogenic E. coli co-displaying IL-12 and ADA effectively reprogram the AML TME, enhance NK cell activation, and drive sustained anti-tumor responses. This live microbial platform offers a safe, localized, and versatile immunotherapy strategy applicable to both hematologic and solid tumors.