p-SCN-Bn-HOPO: A Superior Bifunctional Chelator for 89Zr ImmunoPET

Melissa A. Deri, Shashikanth Ponnala, Paul Kozlowski, Benjamin P. Burton-Pye, Huseyin T. Cicek, Chunhua Hu, Jason S. Lewis, Lynn C. Francesconi
2015 Bioconjugate chemistry  
Zirconium-89 has an ideal half-life for use in antibody-based PET imaging; however, when used with the chelator DFO, there is an accumulation of radioactivity in the bone, suggesting that the 89 Zr 4+ cation is being released in vivo. Therefore, a more robust chelator for 89 Zr could reduce the in vivo release and the dose to nontarget tissues. Evaluation of the ligand 3,4,3-(LI-1,2-HOPO) demonstrated efficient binding of 89 Zr 4+ and high stability; therefore, we developed a bifunctional
more » ... tive, p-SCN-Bn-HOPO, for conjugation to an antibody. A Zr-HOPO crystal structure was obtained showing that the Zr is fully coordinated by the octadentate HOPO ligand, as expected, forming a stable complex. p-SCN-Bn-HOPO was synthesized through a novel pathway. Both p-SCN-Bn-HOPO and p-SCN-Bn-DFO were conjugated to trastuzumab and radiolabeled with 89 Zr. Both complexes labeled efficiently and achieved specific activities of approximately 2 mCi/mg. PET imaging studies in nude mice with BT474 tumors (n = 4) showed good tumor uptake for both compounds, but with a marked decrease in bone uptake for the 89 Zr-HOPO-trastuzumab images. Biodistribution data confirmed the lower bone activity, measuring 17.0%ID/g in the bone at 336 h for 89 Zr-DFO-trastuzumab while 89 Zr-HOPO-trastuzumab only had 2.4%ID/g. We successfully synthesized p-SCN-Bn-HOPO, a bifunctional derivative of 3,4,3-(LI-1,2-HOPO) as a potential chelator for 89 Zr. In vivo studies demonstrate the successful use of 89 Zr-HOPO-trastuzumab to image BT474 breast cancer with low background, good tumor to organ contrast, and, importantly, very low bone uptake. The reduced bone uptake seen with 89 Zr-HOPO-trastuzumab suggests superior stability of the 89 Zr-HOPO complex. Graphical Abstract INTRODUCTION Antibodies possess exquisite specificity and affinity for their antigens, 1 and as a consequence, positron emission tomography (PET) using targeted antibodies is a molecular imaging technique at the forefront of cancer diagnosis and treatment management. 1-6 Zirconium-89 ( 89 Zr), a positron-emitting radionuclide, possesses excellent physical properties for PET imaging when paired with antibodies, namely, an ideal 78.41 h half-life and low energy positron (β avg = 395.5 keV), and is readily attracting attention for this purpose. 7-14 In the past several years, a wide variety of preclinical studies have been published 15-21 and a number of 89 Zr-based antibody imaging agents have been translated into the clinic, including a number of current clinical trials in the US alone. [2] [3] [4] [22] [23] [24] [25] These clinical studies and all preclinical studies use the current standard bifunctional chelator for 89 Zr: desferrioxamine B (DFO). 16 DFO, a natural bacterial siderophore, is a hexadentate ligand with three hydroxamate groups which provide six oxygen donors for metal binding. 26 It possesses an amine tail that can be derivatized for facile conjugation to antibodies and other biomolecular vectors. Although image quality is generally very good, DFO is not the optimal ligand for 89 Zr. This is revealed by the observed uptake of radioactivity in the bone. 7, 16, 27, 28 This uptake is evidence of in vivo release of 89 Zr 4+ from the chelator. When unbound, the osteophilic 89 Zr 4+ cation is readily mineralized into the skeleton. 28,29 This accumulation of 89 Zr 4+ in the bone can dramatically increase radiation dose to the bone marrow, an especially radiosensitive tissue. While the extent of this uptake is less established in the clinic, it is still being investigated and may be of particular concern since 89 Zr-immunoPET agents have found specific use in the detection of bone metastasis. 30 This concern over in vivo stability sparks the need to develop an improved bifunctional chelator for Zr that will significantly improve 89 Zr-antibody PET imaging by providing an Deri et al. improved alternative to DFO, reducing absorbed doses to healthy tissues and therefore safer PET imaging, and enhanced image quality. Recently, there has been a surge of interest in the development of an alternative chelator for 89 Zr 4+ to replace DFO, with several novel ligand systems being reported within the past year or so (Figure 1) . [31] [32] [33] [34] [35] [36] [37] While multiple studies demonstrate the issue of bone uptake seen with 89 Zr-DFO complexes and stress the need for improved chelators, 16, 27, 28, 34 the first investigation toward designing a better chelator of Zr 4+ came from Guérard et al. 38 This work examined the coordination chemistry of the Zr 4+ cation and confirmed the advantage of an octa-coordinate zirconium complex as Zr 4+ was shown to preferentially form complexes with eight oxygen donors contained within four bidentate hydroxamate groups. This study opened the door for the investigation of octadentate ligands to replace the hexadentate DFO chelator with the goal of improving in vivo stability. Thus, far, however, there has been no reporting of a new ligand for 89 Zr 4+ that has been demonstrated to be viable in vivo for a sufficient length of time for antibody imaging. Several potential ligands require additional development while others simply require further evaluation. Herein, we present the first successful demonstration of an alternative chelator for 89 Zr that includes PET imaging and biodistribution data that shows improved stability over DFO across a period of several days in vivo. We investigated the potential of a nonhydroxamate-based ligand-3,4,3-(LI-1,2-HOPO) or HOPO-which has four 1,2-hydroxypyridinone groups for metal binding and comes from the actinide sequestration literature. 39 As we postulated, the HOPO ligand labeled efficiently and 89 Zr-HOPO exhibited equal or superior stability compared to 89 Zr-DFO in all chemical and biological assays. 34 Not only did the 3,4,3-(LI-1,2-HOPO) ligand show tremendous promise in our preliminary evaluation, but even more recently, stability constants for Zr-HOPO were determined to be on the order of log β = 43, the highest recorded for any Zr complex which attests to the superior stability. 40 Therefore, we endeavored to develop a bifunctional variant of the HOPO ligand for further evaluation and application in antibodybased PET imaging. The result of this venture is the bifunctional chelator: p-SCN-Bn-HOPO ( Figure 2 ). This molecule is the HOPO ligand with a para-benzyl-isothiocyanate pendant arm added to one of the secondary amides in order to be directly comparable with the currently most used bifunctional chelator: p-SCN-Bn-DFO ( Figure 2 ). We also report the crystal structure of Zr-HOPO which corroborates the high stability.
doi:10.1021/acs.bioconjchem.5b00572 pmid:26550847 pmcid:PMC4962612 fatcat:prfuk5wdtvf6ja6hc3d67jq4fm