Isolamento e caratterizzazione di anticorpi monoclonali anti-ICOSL

Bone is a connective tissue that is continuously remodeled by the interaction between the cells of the immune system and the cells of the bone. In particular, the two main actors are osteoblasts (responsible for forming new bone) and osteoblasts (responsible for bone resorption). To maintain tissue homeostasis, these two cells' activity must be balanced. When the balance is hampered, pathologies like osteoporosis can occur. It is characterized by excessive bone resorption due to the non-regulation of osteoclastic activity. Osteoporosis is characterized by a loss of bone density, resulting in an increased risk of fractures. To date, there are several therapies for treating the disease, but they are not definitive and have different side effects. Recently, a new signaling pathway involved in the remodeling process has been studied: ICOS / ICOSL. In particular, it has been seen that by treating the osteoclasts with a recombinant molecule called ICOS-Fc (created by Novaicos), it is possible to trigger ICOSL on the surface of the osteoclasts, with consequent inhibition of their activity and maturation both in vitro and in vivo tests. In collaboration with Novaicos, the project aims to replace the ICOS-Fc recombinant molecule with a monoclonal antibody capable of binding ICOSL on osteoclasts, inhibiting their maturation and activity. To do this, in vitro antibody isolation technologies were used. In particular, a combination of phage and yeast visualization was used to combine the positive aspects of the two techniques and reduce their disadvantages. It was possible to exploit the ability to form large libraries of antibodies via phage display and to exploit post-translational modifications and selection processes carried out in real-time via yeast display. Through the phage display technique, it was possible to isolate, starting from a naïve library of antibody fragments, to isolate a monoclonal antibody called G10. The isolated clone showed specific binding to the ICOSL molecule and also good affinity (calculated by BLI: Biolayer interferometry). Furthermore, to better understand the ability of the clone to bind ICOSL in its native conformation, flow cytometric analyzes were performed on different ICOSL + cell types. From these experiments, it was shown that the clone recognizes ICOSL on the cell surface. Given the promising characteristics of this clone, it was decided to further improve the affinity through the 'Affinity Maturation' technique. To do this, the heavy chain variable region (VH) of clone G10 was kept constant and subsequently randomly associated with a library of VL (light chain variable region). The new library was cloned into a vector that allows the expression of the antibody fragment on the yeast surface. Indeed, the yeast display technique was used to isolate clones with improved affinity. Through FACS, it was possible to select the clones with the best affinity in real-time. This led to the isolation of 4 clones. The ‘Affinity maturation' success was verified by calculating the dissociation constant kD (BLI) for all clones. All new clones have a lower kD than the parental clone G10, demonstrating the technique's success. Clone G10 was also characterized as inhibiting osteoclast maturation and activity. Tests conducted in collaboration with Novaicos showed a behavior of clone G10 similar to that of ICOS-Fc. Clone G10 appears to inhibit osteoclastogenesis and, therefore, the maturation of osteoclasts. These data are also confirmed by the reduction of the expression of genes crucial for the development of osteoclasts. The reduction of osteoclastic activity was assessed by quantifying the TRAP enzyme within the osteoclasts treated with the G10 antibody. The data showed a decrease of TRAP protein in G10-treated osteoclasts comparable to that of ICOS-Fc-treated osteoclasts.