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

Area of Science:

• Oncology
• Biotechnology
• Immunology

Background:

• Mesothelin is a tumor-associated antigen highly expressed in various cancers, making it a prime target for diagnosis and therapy.
• Nanobodies offer advantages like tumor penetration and low immunogenicity for targeted cancer treatments.

Purpose of the Study:

• To engineer and evaluate novel anti-mesothelin nanobody constructs for improved cancer theranostics.
• To assess the impact of nanobody format, valency, molecular weight, and half-life extension on tumor targeting and imaging.

Main Methods:

• Engineered six anti-mesothelin nanobody constructs with varying formats, valencies, and half-life extensions (albumin-binding or Fc-fusion).
• Labeled constructs with near-infrared dye for functional evaluation.
• Assessed binding affinity, in vitro 3D tumor penetration, and in vivo xenograft mouse models.

Main Results:

• Functional affinity enhancement varied with molecule format and tumor cell type.
• Bivalency compensated for size limitations below a certain molecular threshold.
• A flexible "pearl necklace" architecture proved optimal for short-term imaging.
• Half-life extension modules improved imaging/therapeutic windows, with albumin-binding superior to Fc-fusion for tumor biodistribution.

Conclusions:

• Nanobody design strategies must be context-dependent and target-specific for optimal anti-mesothelin theranostics.
• Balancing specificity and biodistribution is crucial for maximizing efficacy and minimizing adverse effects.