Tripodal tris(hydroxypyridinone) ligands for immunoconjugate PET imaging with 89Zr4+: comparison with desferrioxamine-B

Abstract

Due to its long half-life (78 h) and decay properties (77% electron capture, 23% β⁺, E_max = 897 keV, E_av = 397 keV, Eγ = 909 keV, Iγ = 100%) ⁸⁹Zr 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 ⁸⁹Zr⁴⁺ because the radiolabeling of the resulting immunoconjugates is rapid under mild conditions. ⁸⁹Zr-DFO complexes are reportedly stable in vitro but there is evidence that ⁸⁹Zr⁴⁺ is released in vivo, and subsequently taken up by the skeleton. We have evaluated a novel tripodal tris(hydroxypyridinone) chelator, H₃CP256 and its bifunctional maleimide derivative, H₃YM103, for coordination of Zr⁴⁺ and compared the NMR spectra, and the ⁸⁹Zr⁴⁺ radiolabeling, antibody conjugation, serum stability and in vivo distribution of radiolabelled immunoconjugates with those of H₃DFO and its analogues. H₃CP256 coordinates ⁸⁹Zr⁴⁺ at carrier-free concentrations forming [⁸⁹Zr(CP256)]⁺. Both H₃DFO and H₃CP256 were efficiently radiolabelled using [⁸⁹Zr(C₂O₄)₄]⁴⁻ at ambient temperature in quantitative yield at pH 6–7 at millimolar concentrations of chelator. Competition experiments demonstrate that ⁸⁹Zr⁴⁺ dissociates from [⁸⁹Zr(DFO)]⁺ in the presence of one equivalent of H₃CP256 (relative to H₃DFO) at pH 6–7, resulting largely in [⁸⁹Zr(CP256)]⁺. To assess the stability of H₃DFO and H₃YM103 immunoconjugates radiolabelled with ⁸⁹Zr, maleimide derivatives of the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side chains. Both immunoconjugates were labelled with ⁸⁹Zr⁴⁺ 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 ⁸⁹Zr⁴⁺ 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 ⁸⁹Zr activity becomes associated with bone in animals administered ⁸⁹Zr-DFO-trastuzumab over the course of 7 days. The tris(hydroxypyridinone) chelator, H₃CP256, coordinates ⁸⁹Zr⁴⁺ rapidly under mild conditions, but the ⁸⁹Zr-labelled immunoconjugate, ⁸⁹Zr-YM103-trastuzumab was observed to release appreciable amounts of ⁸⁹Zr⁴⁺in vivo, demonstrating inferior stability when compared with ⁸⁹Zr-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.