Thiol-mediated Uptake of a Cysteine-containing Nanobody for Anti-Cancer Drug Delivery

Abstract

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Only a few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that recognizes B cell lymphoma in a thiol-dependent manner. CB2 binding strictly requires the presence of a non-conserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 in a thiol-mediated manner such that the nanobody can be used to deliver cytotoxic agents. Hence, surface thiols can be used as lymphoma biomarkers and targeted by thiol-binding nanobodies. Functionalization of internalizable CB2 is the basis for a range of diagnostic and therapeutic applications of this thiol-binding nanobody.TOC GraphicSynopsisNanobody CB2 specifically binds and internalizes into B cell lymphoma via thiol-based interactions. Functionalized CB2 can be used for complement recruitment or drug delivery to lymphoma cells.

Inclusive Pattern Generation Protocols to Decode Thiol-Mediated Uptake

“…1 It is understood that the TMU can operate in combination with different uptake mechanisms. Examples exist for membrane fusion, 24 including the first observation during viral entry, 36 endocytosis, 12,13,15,18,20,22,35 and mostly direct penetration through the plasma membrane. 1 Accounting also for most endosomal escape, the problematic step during endocytosis, direct penetration, has attracted most attention for the cytosolic delivery of SOIs by TMU 1 (the shift from endosomal capture to cytosolic delivery upon activation of TMU has been exemplified with OPS 22 ).…”