Targeted Alpha Therapy for the treatment of multiple myeloma using CD38-targeting sdAb 2F8

Radioligand therapy stands as a promising avenue in the realm of cancer treatment. Targeted alpha therapy (TAT) holds promise due to its ability to selectively administer cytotoxic alpha radiation to tumor cells without affecting neighboring healthy tissues1. This technique exploits the utilization of a carrier molecule, labeled with a therapeutic radionuclide via a chelator, to achieve its targeted delivery. Single-domain antigen-binding fragments (sdAbs), obtained from heavy-chain antibodies of Camelidae species, possess optimal pharmacokinetic attributes owing to their size (approximately 15 kDa) and high affinity for their intended targets2-3. Consequently, they hold promise as carrier molecules for TAT4. This project turns around developing an sdAb-based radiopharmaceutical for TAT treatment of multiple myeloma by radio-labeling the CD38-binding 2F8 sdAb (discovered and developed in our lab)5 with therapeutic radiometals: lutetium-177 (177Lu; emitting β-particles) or actinium-225 (225Ac; emitting α-particles). The whole process involved conjugating the 2F8-sdAb on its 4 lysines with p-SCN-Bn-DOTA (20 equivalents) under pH 8.8-9 conditions in a sodium carbonate buffer, at 4 mg/mL for 3 hours at room temperature. Labeling with 177Lu (25 MBq/μmol) and 225Ac (0.028 MBq/μmol) was executed in a 0.25 M ammonium acetate buffer at pH 5.2, with temperatures set at 50°C and 55°C, respectively. Radiochemical purity (RCP) assessment was conducted through radio-instant thin-layer chromatography (iTLC) and HPLC radio-size-exclusion chromatography (SEC). Evaluation of median effective concentration (EC50) and internalization degree was performed on RPMI CD38+ tumor cells. The conjugation with DOTA yielded an average of 0.4 to 1.4 DOTA molecules per sdAb. RCP exceeded 97% after 45 minutes for 177Lu but reached 75% after 1 hour and 30 minutes for 225Ac. Stability tests in human serum revealed that [177Lu]Lu-DOTA-2F8 remained stable (RCP > 90%) for 24 hours. Moreover, it was noted that [177Lu]Lu-DOTA-2F8 was internalized only for the 20% and exhibited an EC50 of 40 nM (N=1). Further optimization is slated for DOTA conjugation and 225Ac labeling. In-depth in vitro and preclinical investigations with [225Ac]Ac-DOTA-2F8 will be conducted and juxtaposed with its 177Lu-labeled counterpart to elucidate the advantages of TAT, potentially positioning this radiopharmaceutical as a pivotal therapeutic asset for upcoming applications.