Validation of Folate Receptor-α Selectivity of 6 <i>S</i> -5-Methyltetrahydrofolate–Based Radioconjugates: A Prerequisite for Therapeutic Application?

The development of folic acid-based radioconjugates has been proposed for imaging and targeted radionuclide therapy of folate receptor-α (FRα)-positive cancer. Although folic acid binds both the tumor-associated FRα and the folate receptor-β (FRβ) expressed on certain blood cells, 6<i>S</i>-5-methyltetrahydrofolate (6<i>S</i>-5-MTHF) was reported to exhibit higher affinity for FRα. The aim of this study was, therefore, to investigate whether previously developed 6<i>S</i>-5-MTHF-based radioconjugates would preferentially bind to FRα. <b>Methods</b>: The 6<i>R</i>- and 6<i>S</i>-5-MTHF-based folate radioconjugates [¹⁷⁷Lu]Lu-RedFol-1, [¹⁷⁷Lu]Lu-RedFol-3, [¹⁷⁷Lu]Lu-RedFol-24, and [¹⁷⁷Lu]Lu-RedFol-25 were evaluated in vitro using transfected Chinese hamster ovary cells to assess their cell uptake and binding affinity to FRα expressed on RT16 cells and to FRβ expressed on D4 cells. Biodistribution and SPECT/CT imaging studies of the 5-MTHF-based radioconjugates were performed in a mouse model bearing both RT16 and D4 xenografts. Immunohistochemical staining was performed on selected patient-derived tissue sections of high-grade serous ovarian carcinoma, kidney, and bone marrow to provide a representative overview of FRα and FRβ expression. <b>Results:</b> The uptake and receptor binding affinity of 6<i>R</i>-5-MTHF-based radioconjugates were in the same range for RT16 and D4 cells, indicating comparable binding to the FRα and FRβ. The 6<i>S</i>-5-MTHF-based radioconjugates showed a 4-6-fold higher uptake in RT16 cells than in D4 cells, which can be ascribed to the 9-29-fold increased binding affinity to FRα than to FRβ. In vivo, the uptake of 6<i>R</i>-5-MTHF-based radioconjugates in RT16 and D4 xenografts reached 55%-73% IA/g and 25%-44% IA/g at 24 h after injection, respectively. The accumulation of 6<i>S</i>-5-MTHF-based radioconjugates in RT16 xenografts was 74%-100% IA/g, which was 9-21-fold higher than the 4%-10% IA/g uptake observed in D4 xenografts. Immunohistochemistry data confirmed high FRα expression in human ovarian cancer tissue sample and the presence of FRα in renal proximal tubules. In contrast, FRα was absent in human bone marrow, where FRβ was substantially expressed. <b>Conclusion:</b> This study confirmed that the use of 6<i>S</i>-5-MTHF-based radioconjugates can enable selective targeting of FRα. The high expression levels of FRα in ovarian cancer but absence in the bone marrow of human tissue samples infer therapeutic and safety benefits of using FRα-selective folate radioconjugates. Measures to reduce renal retention of 6<i>S</i>-5-MTHF radioconjugates may, however, still be necessary.