Camelid single domain antibodies inhibiting neutrophil serine proteases as therapeutic and prognostic agents in the context of inflammatory diseases

Neutrophil serine proteases (NSPs) are released by neutrophils during inflammation. These proteases, including neutrophil elastase (NE), play a major role in the pathogenesis of various chronic inflammatory diseases such as cystic fibrosis and chronic obstructive pulmonary disease. These conditions are characterized by an imbalance between NSPs and their endogenous inhibitors, leading to excessive and uncontrolled proteolytic activity. This disregulation results in detrimental effects such as progressive tissue degradation. NSPs inhibition constitutes therefore a promising therapeutic target. Additionally, NSPs levels in biological fluids such as sputum and bronchoalveolar lavage correlates with the disease severity making them valuable diagnostic and prognostic biomakers. Camelid single-domain antibody fragments (VHHs) offer unique advantages for the development of both therapeutic inhibitors and diagnostic tools due to their small size, high stability and ability to target cryptic epitopes such as enzyme active sites. In this work, we have generated a VHH, referred to as NbE201, competively and specifically inhibiting NE from both human and murine origins. Various strategies have been carried out to enhance its properties. Finally, efforts were undertaken to select VHHs inhibiting other neutrophil proteases. All together, our results highlight the diagnostic and therapeutical potential of VHHs targeting neutrophil proteases in chronic inflammatory diseases.