Nanobody-based targeting of tissue macrophages for inflammation monitoring during experimental arthritis and hepatitis

Currently, there is a great interest in imaging agents that accumulate and are retained in inflammatory foci by specific interaction with inflammatory cells and that could be developed into safe, available radiopharmaceuticals. Macrophages are one of myeloid lineage cells in the innate immune system and are functionally positioned at the junction of innate and adaptive immune defenses. The ability to image the in vivo biodistribution of these macrophages would thus constitute an in vivo sensor for the status of ongoing immune and inflammatory responses. Immunization of Camelidae with antigens allows a straightforward cloning and selection of singledomain antigen-binding recombinant antibody fragments that matured in vivo, called Nanobodies (Nbs). Their high robustness and stability at elevated temperature allow the fast and easy labeling of their carboxy-terminal hexahistidine tail with 99mTc by tricarbonyl chemistry at elevated temperature and straightforward application as probes in micro-single photon emission computed tomography (SPECT)/micro-CT (?CT). Using 99mTc-labeled Nbs targeting macrophage mannose receptor (MMR) and human epidermal growth factor receptor 2 (HER2), Cellular and Molecular Immunology lab (CMIM) have shown before that these small (15 kD), high-affinity Nbs are excellent tools for molecular tumor and inflammation imaging. In this study we investigated two receptors with a potential to serve as markers of macrophages for in vivo imaging of arthritis mice model and/or liver hepatitis mice model. We investigated the expression of the markers in different cells/organs by and flow cytometry, fluorescence immunohistochemistry assay and assessed their use in in vivo SPECT/ ?CT of mice with collagen induced arthritis (CIA) and/ or with Concanavalin A (ConA) induced liver acute damage. Rheumatoid arthritis (RA) is a chronic inflammatory disorder of unknown cause that is notorious for the chronic polyarticular synovial inflammation and progressive destruction of affected joints. Beside the development of new therapies, diagnosis and monitoring of RA using non-invasive imaging is also characterized by major improvements over the last ten years. Imaging techniques have evolved to allow more rapid and detailed visualization of the disease process. The goal at this point is the characterization of new molecular markers that are specifically expressed in the inflamed joints can provide a potential of non-invasive the RA lesions. V-set and immunoglobulin domain-containing 4 (Vsig4) was investigated since it was described to be expressed in the synovium of RA patients. Our studies showed when expression was investigated in different organs of mice with CIA, we concluded that Vsig4 was rather specifically expressed in the synovium, in addition to the liver where it is known to be expressed on Kupffer cells. SPECT/CT imaging revealed that 99mTc-NbV4m119 specifically targeted Vsig4+ liver macrophages in naïve wild type but not in Vsig4-/- mice. In CIA mice, Nbs against Vsig4 located to the inflamed joints in SPECT imaging. By performing fine dissection after imaging and fluorescence immunohistochemistry, the signal was shown to originate from the synovial and synovial fluid. 99mTc-NbV4m119 accumulation in arthritic lesions increased according to the severity of the inflammation. In knees of mice with CIA, 99mTc-NbV4m119 was found to accumulate even before the onset of macroscopic clinical symptoms. Yet these results offer perspectives for using Nbs