Engineered *E. coli* Nissle 1917 secreting anti-TNF-α nanobody significantly attenuates murine colitis.

The rising global incidence of inflammatory bowel disease (IBD) necessitates safer, gut-targeted therapies. Current treatments, including systemic anti-tumor necrosis factor-alpha (TNF-α) biologics, often lead to off-target immunosuppression, increased infection risk, and poor mucosal bioavailability. Engineered probiotic-based live biotherapeutics offer a compelling alternative by enabling localized drug production within the inflamed intestine, potentially overcoming the limitations of systemic therapies and improving the therapeutic index for IBD patients.

Researchers engineered Escherichia coli Nissle 1917 (EcN) to secrete the anti-TNF-α nanobody MT1, creating the single-strain platform EcN-MT1. Five signal peptides were screened, with α-hemolysin (HlyA) selected for optimal secretion. Plasmid-based and CRISPR-Cas9 chromosomal integration strategies were compared. Binding affinity and anti-inflammatory activity were assessed via ELISA and in LPS-stimulated RAW264.7 macrophages. Therapeutic efficacy was evaluated in a dextran sulfate sodium (DSS)-induced murine colitis model, with oral administration of EcN-MT1. Primary endpoints included body weight, disease activity index (DAI), colon length, histopathology, colonic pro-inflammatory cytokines, and 16S rRNA gut microbiota profiling.

Among the tested signal peptides, α-hemolysin (HlyA) achieved the highest secretion of the nanobody, reaching 4.6 mg/L. The plasmid-based strain markedly outperformed genomic integrants without impairing bacterial growth. Structural modeling and molecular dynamics simulations confirmed stable complementarity-determining regions (CDR)-mediated binding of MT1 to murine TNF-α. This was consistent with a high binding affinity of EC50 27.9 nM and potent suppression of LPS-induced mRNA expression of Tnf and interleukin-1β (Il1b) in macrophages. In the DSS-induced murine colitis model, oral administration of EcN-MT1 significantly attenuated weight loss, improved DAI scores, and preserved colon length. Histopathological analysis revealed reduced mucosal ulceration, crypt loss, and immune cell infiltration. > Oral administration of EcN-MT1 significantly attenuated weight loss, improved DAI scores, and preserved colon length in the DSS-induced murine colitis model.