Anti-CD38 nanobodies as theranostic agents for multiple myeloma

Background Theranostic agents include molecules with a combined diagnostic and therapeutic capability and nanobodies (Nbs) are increasingly used in this field. Nbs are single-domain antigen-binding fragments that are derived from Camelidae heavy-chain antibodies and have several advantages such as their small size leading to better tissue penetration, their favourable pharmacological properties and their ability to recognise small, buried epitopes. Methods The current project aims to use nanobodies directed against CD38 as diagnostic and therapeutic tools in the management of multiple myeloma (MM) disease. The differentiation marker CD38 is highly expressed on myeloma cells isolated from patients with a newly diagnosed or a relapsing disease. Our Nb, retained for its affinity, stability, its favorable biodistribution and its capacity to recognize myeloma cells in a xenograft model of MM, was coupled to Indium-111 and Lutetium-177 and intravenously injected in tumor bearing mice to assess its diagnostic and therapeutic value. Tumor development was assessed by bioluminescence. Results The binding capacities of 111In-DTPA-Nb2F8 was verified by saturation binding experiments (using serial dilutions of the conjugated Nb) on CD38+ RPMI-8226 cells and internalisation experiments were performed in order to assess their therapeutic potential. A minor internalisation (about 20% of the initial bound activity) was observed in the first hours and remained stable during 24H. Micro-SPECT/CT images of mice bearing CD38+ RPMI-8226 tumors and injected with 111In-DPTA-Nb2F8 showed specific tumor targeting 1 hour and at least until 48 hours after injection with a low background signal already 1 hour post-injection (p.i.), except kidneys. The in and ex vivo biodistribution data revealed uptake values in tumor of 3.1% IA/g at 1h post injection, while the uptake of a labelled control Nb was low 0.54% IA/g confirming the specific targeting of our anti-CD38 Nb. Similar biodistributions were found with 177Lu-DTPA-Nb2F8. In order to assess the therapeutic potential of this nanobody, mice with subcutaneously injected RPMI-8226 cells were randomly categorised into 3 groups (n=10). Mice in each group received 3 consecutive intravenous administrations of a high (17.5MBq) or a low radioactive dose (8.6MBq) 177Lu-DTPA-2F8 or an equal volume of vehicle solution. A dose-dependent regression was observed for both therapeutic regimens, which translated in a prolonged median survival from 43 days for vehicle-treated mice, compared to 62 days (p=0.027) for the low and 65 days for the high (p=0.0007) radioactive dose regimen respectively. Conclusions In conclusion, this is the first report on CD38-binding Nbs used for the identification and treatment of MM cells by conjugating them to diagnostic and therapeutic isotopes.