Design and standardization of the technology for the production of ready-to-use kits for conjugated antibody Daratumumab and protein FAPI-based radiopharmaceuticals, intended for labelling with Lu-177- proof of concept for comparative oncology in the

The scientific scope of our group for this project is to design, optimize, and evaluate ready-to-use kits for selective tumor targeting, labeled with the therapeutic radionuclide Lutetium-177 (177Lu). The focus lies on two vector molecules: the mAb antibody Daratumumab, which targets the CD38 antigen, and Fibroblast Activation Protein Inhibitor (FAPI-04) – a small peptide-based molecule designed to target FAP. Following the work planned within this CRP, we defined and evaluated the optimal conditions for the conjugation reaction using three bifunctional chelators—p-SCN-Bn-DOTA, DOTA-NHS ester, and p-SCN-Bn-1B4M-DTPA to achieve efficient labeling with 177Lu. The initial conjugation reactions were performed in phosphate buffer at pH 8.0 and 4°C, overnight, followed by optimization using carbonate buffer at pH 8.5 and 37°C for 1,5 hours, with varying molar excesses of chelators. Among the tested experimental sets, DOTA-SCN demonstrated the best conjugation efficiency and radiochemical stability, confirmed by MALDI-TOF and iTLC analysis. Subsequent work focused on the development of freeze-drying protocols to obtain stable and easily reconstituted formulations. Two series of samples were prepared using saline and ultrapure water as solvents, each containing different excipient compositions: mannitol, sucrose, phosphate buffer, and polysorbate 20. Based on analytical characterization—reconstitution time, residual water content, SE-HPLC for purity, electrophoresis for structural characterization, and radiochemical purity—the saline-based formulation containing mannitol, sucrose, and polysorbate-20 was selected as the most promising. Differential scanning calorimetry (DSC) and Karl Fischer titration were used to define optimal lyophilization parameters. The optimized formulation showed excellent radiochemical stability and preserved antibody structure after conjugation and lyophilization. Parallel experiments with FAPI-04 confirmed high labeling efficiency (>98%) under mild acidic conditions after heating at 95°C for 15 minutes. Labeling of this small peptide proved simpler and more robust than antibody labeling; however, the lyophilized formulation lacked sufficient labeling stability, indicating a need to improve excipient composition further. Ongoing activities include accelerated and long-term stability testing of the developed kits and refinement of the freeze-drying process to improve product appearance and reduce residual moisture. This work establishes a foundation for standardized ¹⁷⁷Lu-based kit technology applicable to both antibody and peptide-based radiopharmaceuticals.