In vivo validation of a novel anti-mesothelin nanobody for early detection of mesothelin-expressing cancers

Abstract

Abstract Antibodies and antibody fragments are widely used in oncology for nanotechnology-based diagnostic, therapeutic, and prognostic assays. Recombinant antibodies that recognize mesothelin, a differentially expressed cancer biomarker with limited normal expression that is upregulated in a variety of epithelial tumors, are important for developing next generation diagnostic or therapeutic platforms and immunosensors because of the flexibility to incorporate various tags or functional groups for site-specific and oriented attachment of antibody fragments to surfaces, labeling reagents or therapeutics. We exploited the inherent stability of camelid heavy chain to develop a site-specific biotinylated, flag-tagged nanobody that recognizes human and mouse mesothelin. We previously published the in vitro characterization of the anti-mesothelin nanobody G3a and then we sought to address whether G3a nanobody could detect mesothelin-expressing tumors in syngeneic and xenograft mouse models of ovarian or lung cancers. Tumor cells were injected subcutaneously (A1847), IV (EKVX, H460, A549), IP or intra ovary (Luc-ID8), and detected at different time points by in vivo imaging (IVIS) or by magnetic resonance imaging (MRI). For in vivo imaging, G3a nanobody was coupled to fluorescently-labelled streptavidin (IRB680W); for MRI, G3a was coupled to avidin-coated magnetic beads. We found that G3a nanobody generated robust and highly mesothelin-specific signals that permitted the detection of very low tumor burden barely detectable by luciferin. Our results indicate that G3a is a valuable reagent for early detection of mesothelin-expressing cancers, as well as a promising delivery agent for imaging and therapeutic conjugates.