Rapid Visualization of Human Tumor Xenografts through Optical Imaging with a Near-Infrared Fluorescent Anti–Epidermal Growth Factor Receptor Nanobody

Abstract

Given that overexpression of the epidermal growth factor receptor (EGFR) is found in many types of human epithelial cancers, noninvasive molecular imaging of this receptor is of great interest. A number of studies have employed monoclonal antibodies as probes; however, their characteristic long half-life in the bloodstream has encouraged the development of smaller probes. In this study, an anti-EGFR nanobody-based probe was developed and tested in comparison with cetuximab for application in optical molecular imaging. To this aim, the anti-EGFR nanobody 7D12 and cetuximab were conjugated to the near-infrared fluorophore IRDye800CW. 7D12-IR allowed the visualization of tumors as early as 30 minutes postinjection, whereas with cetuximab-IR, no signal above background was observed at the tumor site. Quantification of the IR-conjugated proteins in the tumors revealed ≈ 17% of injected dose per gram 2 hours after injection of 7D12-IR, which was significantly higher than the tumor uptake obtained 24 hours after injection of cetuximab-IR. This difference is associated with the superior penetration and distribution of 7D12-IR within the tumor. These results demonstrate that this anti-EGFR nanobody conjugated to the NIR fluorophore has excellent properties for rapid preclinical optical imaging, which holds promise for its future use as a complementary diagnostic tool in humans.

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

“…Tumors were recovered after both 3 and 48 hours, sectioned along the median axis to have access to the inner section, and the emitted infrared fluorescence was recorded ( Figure 6). The mass (80 kDa) of reconstituted IgG-like Abs impairs direct kidney filtration and the rapid clearence specific of monovalent VHHs (14 kDa) [5,30]. Consequently, C8-Fc accumulated in the tumor tissues with the same kinetic of the positive control trastuzumab.…”

“…Although IgG antibodies recovered from hybridoma cells still dominate the field, alternative formats know a constantly increasing success and recombinant Fab, single-chain (scFv) and single-domain (VHH) antibodies have demonstrated their reliability in basic research as well as in clinical applications [1,2]. In the case of VHHs, their reduced mass is a decisive advantage to obtain faster clearance for in vivo imaging, a better penetration in solid tumors and even the permeation across the blood brain barrier [3][4][5]. The in vitro selection allows for the isolation of binders for toxic or scarcely antigenic targets as well as for epitopes correlated to specific functions.…”

Synthesis, Functionalization, and Design of Magnetic Nanoparticles for Theranostic Applications

“…[517] Specifically, nanbobodies have been used as antagonistic drugs [518] and targeting agents of effector domains [519] in cancer immunotherapy. On the other hand, several molecular imaging modalities of SPECT [520] and PET, [521] optical imaging, [522] and ultrasound imaging [523] have been actively directed toward the target by means of nanobodies conjugated to them. Additionally, grafting nanobodies on nanoparticles have shown a considerable potential for increasing their affinity toward the target.…”

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…Given the high stability of nanobodies, a large number of labelling strategies are possible including the use of radionulei [183], near-infrared fluorophores [184] and fluorescent proteins [182]. Nanobodies fused to a fluorescent protein (e.g.…”