Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

Evidence for an active fibrinolytic system in normal human bone marrow.

Normal human bone marrow from patients undergoing heart surgery was analysed quantitatively for components of the fibrinolytic system, using functional and immunological assays. Marrow was found to contain considerable fibrinolytic activity, reflecting high levels of t-PA (tissue-type plasminogen activator). The t-PA was in an active form, despite the presence of the inhibitors PAI-1 and PAI-2. Plasminogen and alpha2-antiplasmin (alpha2-AP) were also present in marrow. The balance of proteases and inhibitors differed dramatically from that observed in plasma, with higher levels of t-PA, PAI-1 and PAI-2, and lower levels of u-PA (urokinase), plasminogen, alpha2-AP and t-PA-PAI-1 complex in bone marrow, and resulted in favourable conditions for fibrinolysis. The presence of plasmin-alpha2-AP complex at concentrations of the same order of magnitude as total plasminogen and alpha2-AP demonstrated that active generation of plasmin was indeed occurring. A role for the active fibrinolytic system in normal human bone marrow may be the removal of unnecessary fibrin deposits formed in the cavities of the marrow, in order to maintain flow through this tissue.