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Description
Radioimmunoconjugates combine the targeting specificity of monoclonal antibodies with the therapeutic effects of radionuclides. Terbium 161 has emerged as a promising candidate owing to its favorable decay properties, including β⁻ emission and conversion/Auger electrons.
The monoclonal antibodies used in this study were rituximab and bevacizumab. Rituximab is a chimeric monoclonal antibody directed against the CD20 antigen expressed on the surface of B lymphocytes and is established in the treatment of B‑cell malignancies, including non‑Hodgkin lymphoma and chronic lymphocytic leukemia. Bevacizumab is a humanized monoclonal antibody targeting vascular endothelial growth factor A (VEGF‑A), a principal mediator of angiogenesis, and has been approved for the treatment of several solid tumors, including colorectal, lung, and renal cell carcinoma.
The purified monoclonal antibodies were conjugated with two chelators, NHS-DOTA
and p-SCN-Bn-DOTA, in 0.2M bicarbonate buffer (pH 9,2). The reaction mixture was gently mixed and incubated at 37°C for 4 h. The immunoconjugates were then isolated
by size-exclusion chromatography with the yield of 70 %.
Radiolabeling of the prepared immunoconjugates was conducted in 0.2M acetate buffer under systematically varied experimental conditions. The effect of pH on labelling efficiency was systematically evaluated at pH 5, 6, and 7. Additionally, different molar ratios
of immunoconjugate to radionuclide were examined to assess their impact on radiolabeling yield and efficiency. The kinetics of the radiolabeling process were also investigated
to elucidate the rate of radionuclide incorporation over time. These studies enabled
the optimization of reaction parameters for efficient and reproducible radiolabeling
of the immunoconjugates.
Finally, in vitro stability tests as well as cell internalization study in human ovaria cancer cells (SK-OV-3) were performer for the prepared [161Tb]DOTA-bevacizumab.
The radioimmunoconjugate was incubated in phosphate-buffered saline (PBS), fetal bovine serum (FBS), and human plasma at 37 °C, 21 °C, and 4 °C. The results showed excellent stability, with radiochemical purity remaining above 98% for all tested conditions. Although bevacizumab does not bind directly to the VEGFA receptor, it is internalized into cells via endocytic vesicles (Karpinska et al., 2023). This internalization mechanism was also observed for [161Tb]DOTA-bevacizumab, which was internalized into SK-OV-3 cells within 120 minutes, thereby delivering a radiocytotoxic payload. SPECT/CT imaging of [161Tb]DOTA-bevacizumab biodistribution in mice showed clear uptake in SK-OV-3 tumors 7 d p.i. as well as the antibody retention in heart and large veins mainly. These findings indicate a high degree of stability and suggest strong potential for future in vivo and clinical applications of the radioimmunoconjugate [161Tb]DOTA-bevacizumab.
This work was supported by AZV CR (NU23-08-00214) and CTU in Prague (SGS25/173/OHK4/3T/14).