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.