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Certain cancers overexpress specific receptors that can be targeted by monoclonal antibodies, such as HER2, which is overexpressed in breast cancer and targeted by pertuzumab. Radioligand therapy builds on this concept by combining antibody specificity with radionuclide-mediated cytotoxicity for selective tumor irradiation.
Terbium‑161 has recently emerged as a promising alternative to lutecium‑177, offering enhanced therapeutic efficacy in small tumors due to its substantially higher emission of conversion and Auger electrons per decay.
This study investigates strategies for the conjugation of pertuzumab with bifunctional chelators, comparing lysine-directed nucleophilic substitution using NHS-DOTA with nucleophilic addition mediated by an isothiocyanate functional group (p‑SCN‑Bn‑DOTA). The conjugation reactions resulted in yields of 34 % for NHS-DOTA and 54 % for p-SCN-Bn-DOTA. The average number of DOTA moieties attached per antibody molecule, determined by MALDI-TOF-MS, was 9 and 12 for the respective conjugates. Furthermore, radiolabelling with Tb-161 was examined, and the stability of the resulting immunoconjugates was systematically evaluated.
Both conjugates were labelled with [161Tb]Cl3 in 0.2 M acetate buffer (pH 6). The optimal reaction conditions, including molar ratio of conjugate to radionuclide and reaction time, were determined for each conjugate. The radiolabelling yielded 51 % and 99 % for the NHS-DOTA and p-SCN-Bn-DOTA conjugates, respectively, under optimized conditions.
In vitro stability studies were performed in phosphate-buffered saline (PBS) and fetal bovine serum (FBS) at room temperature and at 4 °C over one half-life of the radionuclide. The results demonstrate good stability of both DOTA–pertuzumab radioimmunoconjugates in PBS and FBS at 4 °C, with more than 95% of the activity remaining bound to the protein during the first half-life after radiolabelling. At room temperature, a higher degree of activity release was observed; however, more than 90% of the activity remained associated with the conjugate throughout the monitored period. SPECT/CT imaging of 161Tb-labelled pertuzumab biodistribution in tumor-bearing mice revealed very promising in vivo behaviour with the high accumulation in HER2-positive tumors (SK-OV-3) and no uptake in HER-2 negative tumors (MDA-MB231) 7 d p.i. combined with low accumulation in liver.
These results support the potential of radioimmunotherapy as a targeted therapeutic approach. Radiolabelled DOTA–pertuzumab with Tb‑161 demonstrates suitable stability in vitro, warranting further preclinical investigation. Additional studies are required to determine the clinical relevance of these radioimmunoconjugates.
This work was supported by AZV CR (NU23-08-00214) and CTU in Prague (SGS25/173/OHK4/3T/14).