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

Abstract

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.

vNARs as Neutralizing Intracellular Therapeutic Agents: Glioblastoma as a Target

“…Moreover, specific protein knockouts or the modulation of intracellular targets can be achieved by specific nanobodies, granting them the potential to act as prospective therapeutics [ 70 ]. The intrabody strategy has been employed effectively for oncologic targets such as Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor Receptor 2 (VEGF-R2) [ 25 , 71 , 72 , 73 ]. Single-chain antibody fragments (scFv) are the most prevalent format for intrabodies due to the simplicity of their relative expression and intracellular stability compared to full-length IgG antibodies [ 25 , 71 , 73 ].…”

“…The intrabody strategy has been employed effectively for oncologic targets such as Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor Receptor 2 (VEGF-R2) [ 25 , 71 , 72 , 73 ]. Single-chain antibody fragments (scFv) are the most prevalent format for intrabodies due to the simplicity of their relative expression and intracellular stability compared to full-length IgG antibodies [ 25 , 71 , 73 ]. Nevertheless, the ability of scFvs to endure the harsh pH and proteolytic activity within the lysosome represents an obstacle.…”

“…Thus, a more robust biological scaffold is needed. Single domains such as VHH and vNAR represent a promising alternative due to their unique structural features and stability [ 70 , 71 ]. Diverse receptors and transcription factors can be targeted by nanobodies, hindering dysregulated signaling pathways that lead to the unrestrained growth and migration of cancer cells [ 70 ].…”

“…Treatment with vNAR clones attenuated the invasive nature of the 251-cell line across an ECM mimetic matrix assay. [ 71 ] Nb64 VHH α-actinin-4 (ACTN4) Prostate cancer PI3K/AKT-driven signaling pathways interfere by targeting an actin-binding protein, α-actinin-4 (ACTN4). Intracellular expression of Nb64 hampered proliferation, migration, and invasion in prostate cancer cell lines.…”