Head-to-head comparison of DFO* and DFO chelators: selection of the best candidate for clinical ⁸⁹Zr-immuno-PET.

Abstract

Almost all radiolabellings of antibodies with ⁸⁹Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of ⁸⁹Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable ⁸⁹Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with ⁸⁹Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [⁸⁹Zr]Zr-DFO*-NCS and [⁸⁹Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. [⁸⁹Zr]Zr-DFO*-NCS-trastuzumab and [⁸⁹Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [⁸⁹Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates. DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical ⁸⁹Zr-immuno-PET.