H(6)phospa-trastuzumab: bifunctional methylenephosphonate-based chelator with Zr-89, In-111 and Lu-177

The acyclic chelator H(6)phospa and the bifunctional derivative p-SCN-Bn-H(6)phospa have been synthesized using nosyl protection chemistry and evaluated with Zr-89, In-111, and Lu-177. The p-SCN-Bn-H(6)phospa 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 H(6)phospa-trastuzumab immunoconjugate was achieved with In-111 in 70-90% yields at room temperature in 30 minutes, while Lu-177 under the same conditions produced more inconsistent yields of 40-80%. Stability experiments in human serum revealed the In-111-phospa-trastuzumab complex to be 52.0 +/- 5.3% intact after 5 days at 37 degrees C, while the Lu-177-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 In-111-phospa-trastuzumab successfully identified and delineated small (similar to 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 Lu-177-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 H(6)phospa in transient lanthanide chelation for bone-delivery. Radiolabeling with Zr-89 was attempted, but even with elevated temperatures of 37 degrees C, the maximum observed radiometal incorporation over 18 hours was 12%. It can be concluded from this work that H(6)phospa is not superior to the previously studied H(4)octapa for use with In-111 and Lu-177, but improvements in Zr-89 radiolabeling were observed over H(4)octapa, suggesting H(6)phospa to be an excellent starting point for elaboration of Zr-89-based radiopharmaceutical development. To our knowledge, H(6)phospa is the best desferrioxamine alternative for Zr-89 radiolabeling to be studied to date.