Direct comparison of the in vitro and in vivo stability of DFO, DFO* and DFOcyclo* for ⁸⁹Zr-immunoPET.

Abstract

Currently, the most commonly used chelator for labelling antibodies with ⁸⁹Zr for immunoPET is desferrioxamine B (DFO). However, preclinical studies have shown that the limited in vivo stability of the ⁸⁹Zr-DFO complex results in release of ⁸⁹Zr, which accumulates in mineral bone. Here we report a novel chelator DFOcyclo*, a preorganized extended DFO derivative that enables octacoordination of the ⁸⁹Zr radiometal. The aim was to compare the in vitro and in vivo stability of [⁸⁹Zr]Zr-DFOcyclo*, [⁸⁹Zr]Zr-DFO* and [⁸⁹Zr]Zr-DFO. The stability of ⁸⁹Zr-labelled chelators alone and after conjugation to trastuzumab was evaluated in human plasma and PBS, and in the presence of excess EDTA or DFO. The immunoreactive fraction, IC₅₀, and internalization rate of the conjugates were evaluated using HER2-expressing SKOV-3 cells. The in vivo distribution was investigated in mice with subcutaneous HER2⁺ SKOV-3 or HER2^- MDA-MB-231 xenografts by PET/CT imaging and quantitative ex vivo tissue analyses 7 days after injection. ⁸⁹Zr-labelled DFO, DFO* and DFOcyclo* were stable in human plasma for up to 7 days. In competition with EDTA, DFO* and DFOcyclo* showed higher stability than DFO. In competition with excess DFO, DFOcyclo*-trastuzumab was significantly more stable than the corresponding DFO and DFO* conjugates (p < 0.001). Cell binding and internalization were similar for the three conjugates. In in vivo studies, HER2⁺ SKOV-3 tumour-bearing mice showed significantly lower bone uptake (p < 0.001) 168 h after injection with [⁸⁹Zr]Zr-DFOcyclo*-trastuzumab (femur 1.5 ± 0.3%ID/g, knee 2.1 ± 0.4%ID/g) or [⁸⁹Zr]Zr-DFO*-trastuzumab (femur 2.0 ± 0.3%ID/g, knee 2.68 ± 0.4%ID/g) than after injection with [⁸⁹Zr]Zr-DFO-trastuzumab (femur 4.5 ± 0.6%ID/g, knee 7.8 ± 0.6%ID/g). Blood levels, tumour uptake and uptake in other organs were not significantly different at 168 h after injection. HER2^- MDA-MB-231 tumour-bearing mice showed significantly lower tumour uptake (p < 0.001) after injection with [⁸⁹Zr]Zr-DFOcyclo*-trastuzumab (16.2 ± 10.1%ID/g) and [⁸⁹Zr]Zr-DFO-trastuzumab (19.6 ± 3.2%ID/g) than HER2⁺ SKOV-3 tumour-bearing mice (72.1 ± 14.6%ID/g and 93.1 ± 20.9%ID/g, respectively), while bone uptake was similar. ⁸⁹Zr-labelled DFOcyclo* and DFOcyclo*-trastuzumab showed higher in vitro and in vivo stability than the current commonly used ⁸⁹Zr-DFO-trastuzumab. DFOcyclo* is a promising candidate to become the new clinically used standard chelator for ⁸⁹Zr immunoPET.