Development and testing of anti-eNAMPT neutralizing monoclonal antibodies

Nicotinamide phosphoribosyltransferase (NAMPT) is an intracellular enzyme that plays an essential role in mammalian cell metabolism by participating to NAD+ production. Through a mechanism not yet deciphered, NAMPT is secreted in the extracellular space and is therefore referred to as extracellular NAMPT (eNAMPT). In the extracellular milieu, eNAMPT has been shown to execute multiple functions, from adipokine to pro-inflammatory activities, likely by interacting with cell surface receptors. Circulating eNAMPT levels have been associated with various inflammatory conditions, such as cancer, suggesting a potential for this protein as a diagnostic tool. Furthermore, an active role for eNAMPT in cancer pathogenesis has also been proposed, based on studies demonstrating that eNAMPT supports tumor cell proliferation, stimulates tumor invasion and metastasis and promotes immunosupressive conditions. However, eNAMPT pro-tumoral functions were shown to be independent of enzymatic activity, indicating that they cannot be counteracted by chemical inhibitors. A new approach, using monoclonal antibodies, has therefore been suggested to neutralize eNAMPT extracellular functions. My PhD project aimed to develop and test novel anti-eNAMPT neutralizing monoclonal antibodies (mAbs). Firstly, I generated 81 specific, fully human anti-eNAMPT mAbs using a phage display platform. Characterization of these antibodies revealed that they exhibited different binding characteristics and targeted different regions on eNAMPT. In addition, several of them showed a high affinity for eNAMPT. Secondly, I sought to assess the neutralizing properties of these anti-eNAMPT mAbs in relevant in vitro binding and functional assays. However, I was unable to convincingly demonstrate eNAMPT interaction with its putative receptors, nor the pro-tumoral, pro-chemotactic and pro-inflammatory effects of eNAMPT. As a result, I could not evaluate the blocking abilities of the generated eNAMPT binding antibodies.