Nanobodies as Versatile Tool for Multiscale Imaging Modalities

Abstract

Molecular imaging is constantly growing in different areas of preclinical biomedical research. Several imaging methods have been developed and are continuously updated for both in vivo and in vitro applications, in order to increase the information about the structure, localization and function of molecules involved in physiology and disease. Along with these progresses, there is a continuous need for improving labeling strategies. In the last decades, the single domain antigen-binding fragments nanobodies (Nbs) emerged as important molecular imaging probes. Indeed, their small size (~15 kDa), high stability, affinity and modularity represent desirable features for imaging applications, providing higher tissue penetration, rapid targeting, increased spatial resolution and fast clearance. Accordingly, several Nb-based probes have been generated and applied to a variety of imaging modalities, ranging from in vivo and in vitro preclinical imaging to super-resolution microscopy. In this review, we will provide an overview of the state-of-the-art regarding the use of Nbs in several imaging modalities, underlining their extreme versatility and their enormous potential in targeting molecules and cells of interest in both preclinical and clinical studies.

Keywords: imaging; microscopy; nanobody.

Figure 1

Nanobodies (Nbs) as imaging tool for the visualization of different pathological conditions. Nbs have been successfully used for the visualization of a variety of pathologies. Different aspects of neoplastic progression have been evaluated using Nbs targeting tumor cells (EGFR-Nb, HER2-Nb, CAIX-Nb and CEA-Nb), tumor angiogenesis (NJB2-Nb and VCAM-1-Nb), TAM infiltration (MMR-Nb and MHC-II-Nb) or components of immune checkpoints (PD-L1-Nb, CTLA-4-Nb and LAG-3-Nb). Similarly, several inflammatory diseases have been successfully monitored using Nbs, including liver inflammation (Vsig-Nb and ClecF4-Nb), diabetes (DPP6-Nb), rheumatoid arthritis (MMR-Nb, NbV4-Nb and Vsig-Nb) and atherosclerosis (MMR-Nb and VCAM-1-Nb). Moreover, Nbs have been applied for the visualization of brain tumors (EGFR-Nb and IGFBP7-Nb). In addition, due to their small dimension, Nbs more easily cross the blood brain barrier and accumulate into the brain parenchyma (FC5-Nb, FC44-Nb, GFAP-Nb and Aβ-Nb).

Figure 2

Imaging tumor with Nbs. In the last decades, several Nbs have been generated to detect different components and cell populations of the tumor microenvironment in vivo. Nbs directed against HER2, CAIX, EGFR and CEA have been widely used, alone or in combination to visualize neoplastic cells. Tumor angiogenesis and extracellular matrix (ECM) composition have been investigated using anti-VCAM-1 and anti-NJB2 Nbs. Moreover, pro-tumorigenic tumor-associated macrophages (TAMs) have been identified by anti-MMR Nbs, while TAMs associated to anti-tumor response have been detected by anti-MHC-II Nbs. Other Nb-based probes have also been designed to target antigen presenting cells (APCs). Finally, several Nbs have been developed for immune-checkpoint inhibitor, including CTLA-4 and PD-L1. More recently, anti-LAG3 Nbs have been generated to detect tumor-infiltrating lymphocytes.

Figure 3

Nbs minimize the “linkage error.” Due to their smaller dimension compared to conventional Abs, the use of Nbs significantly reduces the distance between the antigen and the fluorophore, thus minimizing the so-called “linkage error.”.