H6phospa-Trastuzumab: BifunctionalMethylenephosphonate-based Chelator with 89Zr 111In and177Lu

摘要

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 that¹¹¹In-phospa-trastuzumab successfully identified and delineated small(~2 mm in diameter) tumors from surrounding tissues, despite visibleuptake in the kidneys and bone due to moderate chelate instability. As predictedfrom stability assays in serum, the ¹⁷⁷Lu-phospa-trastuzumabconjugate served as a negative control and displayed no tumor uptake, with highuptake in bones indicating rapid and complete radiometal dissociation andsuggesting a potential application of H6phospa in transientlanthanide chelation for bone-delivery. Radiolabeling with ⁸⁹Zr wasattempted, but even with elevated temperatures of 37 °C, the maximumobserved radiometal incorporation over 18 hours was 12%. It can beconcluded from this work that H6phospa is not superior to thepreviously studied H4octapa for use with ¹¹¹In and¹⁷⁷Lu, but improvements in ⁸⁹Zr radiolabeling wereobserved over H4octapa, suggesting H6phospa to be anexcellent starting point for elaboration of ⁸⁹Zr-basedradiopharmaceutical development. To our knowledge, H6phospa is thebest desferrioxamine alternative for ⁸⁹Zr radiolabeling to be studiedto date.