Novel bispecific nanobody mitigates experimental intestinal inflammation in mice by targeting TNF‐α and IL‐23p19 bioactivities

Abstract Background Inflammatory bowel diseases (IBDs) pose significant challenges in terms of treatment non‐response, necessitating the development of novel therapeutic approaches. Although biological medicines that target TNF‐α (tumour necrosis factor‐α) have shown clinical success in some IBD patients, a substantial proportion still fails to respond. Methods We designed bispecific nanobodies (BsNbs) with the ability to simultaneously target human macrophage‐expressed membrane TNF‐α (hmTNF‐α) and IL‐23. Additionally, we fused the constant region of human IgG1 Fc (hIgG1 Fc) to BsNb to create BsNb‐Fc. Our study encompassed in vitro and in vivo characterization of BsNb and BsNb‐Fc. Results BsNb‐Fc exhibited an improved serum half‐life, targeting capability and effector function than BsNb. It's demonstrated that BsNb‐Fc exhibited superior anti‐inflammatory effects compared to the anti‐TNF‐α mAb (infliximab, IFX) combined with anti‐IL‐12/IL‐23p40 mAb (ustekinumab, UST) by Transwell co‐culture assays. Notably, in murine models of acute colitis brought on by 2,4,6‐trinitrobenzene sulfonic acid（TNBS) and dextran sulphate sodium (DSS), BsNb‐Fc effectively alleviated colitis severity. Additionally, BsNb‐Fc outperformed the IFX&UST combination in TNBS‐induced colitis, significantly reducing colon inflammation in mice with colitis produced by TNBS and DSS. Conclusion These findings highlight an enhanced efficacy and improved biostability of BsNb‐Fc, suggesting its potential as a promising therapeutic option for IBD patients with insufficient response to TNF‐α inhibition. Key points A bispecific nanobody (BsNb) was created to target TNF‐α and IL‐23p19, exhibiting high affinity and remarkable stability. BsNb‐Fc inhibited the release of cytokines in CD4+T cells during co‐culture experiments. BsNb‐Fc effectively alleviated colitis severity in mouse model with acute colitis induced by DSS or TNBS, outperforming the IFX&UST combination.