89Zr-immuno-PET in translational development of biopharmaceuticals

Abstract

Biologicals gained attention over the past decades thanks to their therapeutic success especially in oncology. Positron Emission Tomography (PET) with Zirconium-89 is attractive thanks to 89Zr half-life (78.41h) matching the biological half-life of monoclonal antibodies (mAbs). We provide an overview of 89Zr-immuno-PET imaging using current and emerging radiolabeling tools and preclinical imaging to facilitate translation to the clinic of new antibody constructs. By improving the radiochemistry toolbox and better understanding quantification using preclinical PET cameras, new opportunities can be translated in the clinic. Chapter 1 introduces the current position of PET and 89Zr-immuno-PET imaging, in the context of biopharmaceuticals development while Chapter 2 gives an overview of current radiolabeling methods of biopharmaceuticals with 89Zr, 64Cu and 68Ga but also less common radiometals: 52Mn, 86Y, 66Ga, 44Sc, and 18F as in [18F]AlF. Chelator-radionuclide pairs and radiolabeling conditions are discussed along with recent preclinical and clinical trends. Chapter 3 evaluates with 89Zr-immuno-PET, CX-2009, a Probody Drug Conjugate (PDC) with a toxic DM4 payload attached. Probody® therapeutics possess antigen binding domains masked by peptide caps, only removed in the tumor environment by tumor-associated proteases, locally overexpressed. Probodies aim at widening the therapeutic window while PDCs at delivering selectively their payload to tumors via widely expressed antigens. We evaluated CX-2009 in CD166-positive lung cancer mice in comparison with its Probody, unmasked antibody drug conjugate, and parental mAb derivatives. Tumor uptake was similar for all constructs 72h p.i. demonstrating that CX-2009 can target CD166-expressing tumors and thus supporting clinical evaluation of Probody® therapeutics. In chapter 4, the novel octadentate chelator DFO* was studied in depth and compared with desferrioxamine (DFO), current standard for 89Zr-immuno-PET, DFOSq, also reported as potential successor of DFO and DFO*Sq included to evaluate the extra hydroxamate or squaramide group contribution to 89Zr complexation. [89Zr]Zr-DFO*-NCS-trastuzumab and [89Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [89Zr]Zr-DFO counterparts. In breast cancer mice, DFO* derivatives were more stable than DFO derivatives especially in bones. DFOSq did not outperform the DFO derivative, suggesting that Sq is not improving in vivo stability. Cetuximab, directed against the Epidermal-Growth-Factor-Receptor was used in xenograft mice and again DFO* was superior over DFO regarding bone uptake. In an intratibial bone metastasis model, [89Zr]Zr-DFO*-trastuzumab, [89Zr]Zr-DFO-trastuzumab, [89Zr]Zr-DFO*-B12 and [89Zr]Zr-DFO-B12 (non-targeting control mAb) were evaluated and the DFO*-conjugate appeared superior over the DFO-conjugate with a tumour-specific signal in bone tumors. Chapter 5, provides in-depth comparison between PET imaging and ex vivo biodistribution quantification. We performed phantom studies with a NanoScan PET/CT and PET/MR with the most used PET radionuclides (11C, 68Ga, 18F and 89Zr). The cameras performed similarly: the highest recovery coefficient being with 18F, followed by 11C and 89Zr and finally 68Ga. Both scanners were evaluated after injection of [18F]FDG and [89Zr]Zr-DFO-NCS-trastuzumab in breast cancer mice and performed equally well regarding tumor quantification with PET-assessed uptake lower than ex vivo values. In brains, [18F]FDG-PET/ex vivo ratios were excellent suggesting that brain is suitable for quantitative imaging of 18F-tracers but not for 89Zr-radiolabeled-mAbs, probably due to poor brain penetration. In kidney and liver more disparities were observed. Preclinical cameras and ex vivo biodistribution quantification, requires fully described standardised protocols for reliability, reproducibility and inter-study comparisons. This research offers a state-of-the-art overview and promising developments regarding 89Zr-immuno-PET imaging. It provides new insights on preclinical studies including quantification with preclinical scanners and new tools for radiolabeling biologicals confirming 89Zr-immuno-PET imaging potential to evaluate new constructs. In vitro and in vivo superiority of the new chelator DFO* over the gold standard for clinical 89Zr-immuno-PET, DFO, was confirmed in various models, especially regarding bone uptake. DFO* is thus considered as the successor of DFO for clinical applications.