Tripodal tris(hydroxypyridinone) ligands for immunoconjugate PET imaging with Zr-89(4+): comparison with desferrioxamine-B

Due to its long half-life (78 h) and decay properties (77% electron capture, 23% beta(+), E-max = 897 keV, E-av = 397 keV, E gamma = 909 keV, I gamma = 100%) Zr-89 is an appealing radionuclide for immunoPET imaging with whole IgG antibodies. Derivatives of the siderophore desferrioxamine-B (H3DFO) are the most widely used bifunctional chelators for coordination of Zr-89(4+) because the radiolabeling of the resulting immunoconjugates is rapid under mild conditions. Zr-89-DFO complexes are reportedly stable in vitro but there is evidence that Zr-89(4+) is released in vivo, and subsequently taken up by the skeleton. We have evaluated a novel tripodal tris(hydroxypyridinone) chelator, H(3)CP256 and its bifunctional maleimide derivative, H(3)YM103, for coordination of Zr4+ and compared the NMR spectra, and the Zr-89(4+) radiolabeling, antibody conjugation, serum stability and in vivo distribution of radiolabelled immunoconjugates with those of H3DFO and its analogues. H(3)CP256 coordinates Zr-89(4+) at carrier-free concentrations forming [Zr-89(CP256)](+). Both H3DFO and H(3)CP256 were efficiently radiolabelled using [Zr-89(C2O4)(4)](4-) at ambient temperature in quantitative yield at pH 6-7 at millimolar concentrations of chelator. Competition experiments demonstrate that Zr-89(4+) dissociates from [Zr-89(DFO)](+) in the presence of one equivalent of H(3)CP256 (relative to H3DFO) at pH 6-7, resulting largely in [Zr-89(CP256)](+). To assess the stability of H3DFO and H(3)YM103 immunoconjugates radiolabelled with Zr-89, maleimide derivatives of the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side chains. Both immunoconjugates were labelled with Zr-89(4+) in >98% yield at high specific activities and the labeled immunoconjugates were stable in serum with respect to dissociation of the radiometal. In vivo studies in mice indicate that Zr-89(4+) dissociates from YM103-trastuzumab with significant amounts of activity becoming associated with bones and joints (25.88 0.58% ID g(-1) 7 days post-injection). In contrast, <8% ID g(-1) of Zr-89 activity becomes associated with bone in animals administered Zr-89-DFO-trastuzumab over the course of 7 days. The tris(hydroxypyridinone) chelator, H(3)CP256, coordinates Zr-89(4+) rapidly under mild conditions, but the Zr-89-labelled immunoconjugate, Zr-89-YM103-trastuzumab was observed to release appreciable amounts of Zr-89(4+) in vivo, demonstrating inferior stability when compared with Zr-89-DFO-trastuzumab. The significantly lower in vivo stability is likely to be a result of lower kinetic stability of the Zr4+ tris(hydroxypyridinone complex) relative to that of DFO and its derivatives.