An Inflammation‐Targeting Engineered Probiotic Escherichia coli Nissle 1917 with High Anti‐TNF‐α Nanobody Secretion Efficacy Alleviates Ulcerative Colitis

Abstract The probiotic Escherichia coli Nissle 1917 (EcN), clinically used for ulcerative colitis (UC) due to its safety and genetic tractability, exhibits limited therapeutic efficacy owing to poor targeting and colonization at inflamed sites. Phosphatidylserine (PS) exposure increases on apoptotic colon epithelial cells during UC progression, suggesting PS‐targeting could enhance EcN localization. Here, EcN is engineered to surface‐display Annexin A5 (ANXA5), a PS‐binding protein, to improve inflammation targeting and colonization. Since TNF‐α drives UC pathogenesis and anti‐TNF‐α biologics face cost and safety limitations, the secretion‐hindering lpp gene is concurrently knocked out in ANXA5‐expressing EcN, creating the inflammation‐targeted, secretion‐enhanced EcN Δlpp ::A5. This base strain is further modified to secrete an anti‐TNF‐α nanobody (aTN), generating EcN Δlpp ::A5‐aTN. Both engineered strains demonstrate significantly stronger colonic colonization versus wild‐type EcN, effectively attenuating oxidative stress‐mediated epithelial apoptosis, restoring mucosal barriers, improving immune‐microbiota balance, and alleviating murine colitis. EcNΔ lpp ::A5‐aTN shows superior efficacy to EcNΔ lpp ::A5. This study develops an engineered EcN system with enhanced targeting, colonization, and secretion. By efficiently delivering anti‐TNF‐α nanobodies, EcNΔ lpp ::A5‐aTN exhibits strong therapeutic potential for UC, overcoming limitations that hinder the clinical application of wild‐type EcN.