Dual-epitope targeting of TSLP to simultaneously block TSLPR and IL-7Rα with a Biparatopic nanobody

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that plays a crucial role in the pathophysiology of asthma, initiating multiple allergic cascade responses. Tezepelumab is the only monoclonal antibody currently approved for marketing, which acts by blocking TSLP binding to TSLPR. However, it is reported that a TSLP trap which simultaneously block TSLP binding with TSLPR and IL-7Rα has better efficiency in the repression of TSLP signal pathway. In this study, we identified two monovalent nanobodies targeting TSLP through immunization of alpacas and phage display screening. They could block TSLP binding with the TSLPR and IL-7Rα receptors, respectively. Then, various protein linkers were designed between these two monovalent nanobodies to form different biparatopic nanobodies. We found that the affinity and biological activity of biparatopic nanobodies are related to the length of the linkers and the tandem order of the nanobodies. We screened a biparatopic nanobody CRNB909, which exhibits extremely high binding affinity for human TSLP with a KD < 1E-12 M, demonstrating approximately 40-fold greater potency compared to the Tezepelumab antibody (KD = 3.98E-11 M). Furthermore, it shows superior functional activity in biological assays, displaying 9.2-fold enhanced inhibition of TSLP-mediated JAK-STAT signaling pathway activation and 4.3-fold greater suppression of TSLP-induced BAF3 cell proliferation relative to Tezepelumab. Finally, CRNB909 was successfully secretory expressed in Komagataella phaffii with reduced production costs. This study provides a biparatopic nanobody that could bind TSLP to simultaneously block TSLPR and IL-7Rα, which might be a promising therapeutant for TSLP related diseases.