H6phospa-trastuzumab: bifunctional methylenephosphonate-based chelator with 89Zr, 111In and 177Lu

Abstract

The acyclic chelator H₆phospa and the bifunctional derivative p-SCN–Bn–H₆phospa have been synthesized using nosyl protection chemistry and evaluated with ⁸⁹Zr, ¹¹¹In, and ¹⁷⁷Lu. The p-SCN–Bn–H₆phospa derivative was successfully conjugated to trastuzumab with isotopic dilution assays indicating 3.3 ± 0.1 chelates per antibody and in vitro cellular binding assays indicating an immunoreactivity value of 97.9 ± 2.6%. Radiolabeling of the H₆phospa-trastuzumab immunoconjugate was achieved with ¹¹¹In in 70–90% yields at room temperature in 30 minutes, while ¹⁷⁷Lu under the same conditions produced more inconsistent yields of 40–80%. Stability experiments in human serum revealed the ¹¹¹In-phospa-trastuzumab complex to be 52.0 ± 5.3% intact after 5 days at 37 °C, while the ¹⁷⁷Lu-phospa-trastuzumab to be only 2.0 ± 0.3% intact. Small animal SPECT/CT imaging using mice bearing subcutaneous SKOV-3 ovarian cancer xenografts was performed, and it was found that ¹¹¹In-phospa-trastuzumab successfully identified and delineated small (∼2 mm in diameter) tumors from surrounding tissues, despite visible uptake in the kidneys and bone due to moderate chelate instability. As predicted from stability assays in serum, the ¹⁷⁷Lu-phospa-trastuzumab conjugate served as a negative control and displayed no tumor uptake, with high uptake in bones indicating rapid and complete radiometal dissociation and suggesting a potential application of H₆phospa in transient lanthanide chelation for bone-delivery. Radiolabeling with ⁸⁹Zr was attempted, but even with elevated temperatures of 37 °C, the maximum observed radiometal incorporation over 18 hours was 12%. It can be concluded from this work that H₆phospa is not superior to the previously studied H₄octapa for use with ¹¹¹In and ¹⁷⁷Lu, but improvements in ⁸⁹Zr radiolabeling were observed over H₄octapa, suggesting H₆phospa to be an excellent starting point for elaboration of ⁸⁹Zr-based radiopharmaceutical development. To our knowledge, H₆phospa is the best desferrioxamine alternative for ⁸⁹Zr radiolabeling to be studied to date.