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

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-alpha (tumour necrosis factor-alpha) 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-alpha (hmTNF-alpha) 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-alpha 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-alpha inhibition.