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

Abstract

Purpose Almost all radiolabellings of antibodies with 89 Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89 Zr 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 89 Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. Methods Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89 Zr 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, [ 89 Zr]Zr-DFO*-NCS and [ 89 Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. Results [ 89 Zr]Zr-DFO*-NCS-trastuzumab and [ 89 Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [ 89 Zr]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. Conclusion DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89 Zr-immuno-PET. Keywords Bone metastasis model. DFO. DFO*. DFOSq. DFO*Sq. 89 Zr-immuno-PET This article is part of the Topical Collection on Preclinical Imaging

The Role of 89Zr-Immuno-PET in Navigating and Derisking the Development of Biopharmaceuticals

“…When comparing the performance of these chelators, it appears that the octadentate analog of DFO consisting of 4 hydroxamate moieties, called DFO* (''DFO star''), is able to solve the instability issues observed with 89 Zr-DFO (Fig. 1) (17). Because the bifunctional variant DFO*-NCS is now commercially available, its translation to the clinic can be expected soon.…”

The Race for Hydroxamate-Based Zirconium-89 Chelators

“…In most cases, their performance is assessed in comparison to the current standard DFO. While this practice is well justified, a direct comparison among the new chelators for the identification of the most promising candidate(s) for clinical translation is, with few exceptions [28,44,45,48], still pending. Along these lines, a standardization of in vitro experiments studying the stability of 89 Zr-complexes would be highly desirable to facilitate a direct comparison of reported data.…”

“…Additionally, in vivo studies on the intratibial BT-474 model showed much better tumor-to-background contrast due to less unspecific accumulation of 89 Zr in bones. The authors concluded that DFO-immunoconjugates are less suitable for the detection of bone metastases and again highlighted the superiority of DFO* derivatives in comparison to the current gold standard DFO [44].…”

“…The most comprehensive in vivo comparison of the chelators DFO-NCS, DFO*-NCS, DFOsqOEt and DFO*sqOEt so far was reported by Chomet et al [44]. The study not only revealed important insights into in vivo stability of the 89 Zr-complexes, but also provided valuable in vitro stability data, which contribute to the understanding of the complexation behavior of the squaramide group.…”

A Semi Rigid Novel Hydroxamate AMPED-Based Ligand for 89Zr PET Imaging

“…In particular, the octadentate version of the DFO chelator, the so-called DFO*, has been developed. Two different functionalized versions of this chelator were synthesized, conjugated with a monoclonal antibody, and tested in vivo showing a lower 89 Zr bone uptake over the DFO conjugate [ 24 ]. Nowadays, the development of radiotracers for PET imaging is focused on the use of antibodies or antibodies fragments as carriers.…”