Optimizing nanobody-based molecules targeting mesothelin: Exploring format, valency, size, and half-life for next-generation cancer theranostics.

Mesothelin is a tumor-associated antigen highly expressed in various cancers, with limited presence in normal tissues, making it an ideal target for cancer diagnosis and therapy. Nanobodies, small single-domain antibody fragments, possess favorable properties such as excellent tumor penetration, low immunogenicity, and versatility in design. This study engineered six anti-mesothelin nanobody-based constructs differing in format, valency, molecular weight, and half-life extension strategies, using either albumin-binding domains or Fc-fusion. All constructs were labeled with a near-infrared dye for functional evaluation. Binding affinity was measured, and tumor penetration was assessed in vitro using three-dimensional tumor models combining cancer cells and fibroblasts, as well as in vivo in xenografted mouse models. Key findings demonstrate that i) the enhancement of functional affinity is influnced by both the molecule format and the tumor cell type, ii) below a certain molecular size, bivalency can compensate size-related limitations, iii) the flexible "pearl necklace" architecture is the most suitable option for achieving optimal short term imaging, and iv) the incorporation of half-life extension modules extends imaging and therapeutic windows, with albumin-binding-based strategies outperforming Fc-fusion in tumor biodistribution, which must be carefully balanced with specificity to maximize efficacy and minimize adverse effects. In conclusion, this study contributes to the development of more effective next-generation anti mesothelin theranostic approaches and highlights that nanobody properties and optimal design strategies may be target specific, emphasizing the need for context-dependent development.