Thiol-Mediated Uptake of a Cysteine-Containing Nanobody for Anticancer 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. Few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that specifically recognizes B cell lymphoma and breast cancer in a thiol-dependent manner. CB2 binding strictly requires the presence of a nonconserved 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 via thiol-mediated endocytosis which can be exploited to deliver cytotoxic agents. CB2 internalization combined with functionalization forms the basis for a wide range of diagnostic and therapeutic applications, rendering thiol-reactive nanobodies promising tools for targeting cancer.

Utilizing fab fragment‐conjugated surface plasmon resonance‐based biosensor for detection of SalmonellaEnteritidis

“…38 Despite the prevalent use of these strategies based on antibody reduction in tumor detection, their application in foodborne bacteria detection remains limited. [39][40][41][42] Given the challenges associated with detecting low-level pathogens, our study introduces a novel approach to Salmonella Enteritidis detection by employing Fab fragmentconjugated SPR-based biosensors. While the detection of Salmonella has been explored in the literature, this work stands out due to the specific use of Fab fragments in conjunction with SPR technology offers improved sensitivity.…”

“…Despite the prevalent use of these strategies based on antibody reduction in tumor detection, their application in foodborne bacteria detection remains limited 39–42 . Given the challenges associated with detecting low‐level pathogens, our study introduces a novel approach to Salmonella Enteritidis detection by employing Fab fragment‐conjugated SPR‐based biosensors.…”

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 ).…”

“…Thiol-mediated uptake (TMU) refers to the cell-penetrating activity acquired by the attachment of thiol-reactive motifs to the substrates of interest (SOI), − such as probes, , drugs, proteins, ,,,− oligonucleotides, − liposomes, ,− quantum dots, nanoparticles, ,, and so on (Figure ). Since this process appears to operate with dynamic covalent exchange reactions with cellular thiols and disulfides, e.g., cysteine and cystine residues, it can be inhibited by other thiol-reactive agents, which is the hallmark of TMU.…”