Optimising Ligand Design For The 89Zr/177Lu Theranostic Pair

Abstract

Theranostic treatment describes the marriage of diagnostic and therapeutic techniques to employ a holistic approach to patient care. Theranostic approaches are increasing in the field of radioimmunotherapy, with a focus on oncological treatments. Techniques such as immunological positron emission tomography (immunoPET) imaging can be used to infer the location of cancers or the presence of cell-surface biomarkers, applicable as drug targets. Radioimmunotherapeutics can then be administered, drawing on this diagnostic information to optimise therapeutic outcomes. Zirconium-89 (89Zr) and lutetium-177 (177Lu) see current use in research as a theranostic pairing for oncological treatment. 89Zr is a β+-emitting radioisotope, with use in immunoPET imaging, while 177Lu is a β--emitting radioisotope with widespread adoption in radioimmunotherapeutic drugs. As both radionuclides are metal ions, an organic ligand is required to chelate them for use. ImmunoPET and radioimmunotherapy both require the use of monoclonal antibodies (mAb) or other targeting motifs and these ligands must possess the potential for further functionalisation to allow conjugation of these entities. Radiometals have particular preferences for coordination, placing further restriction on the design of these ligand chelates for theranostic use. This thesis aimed to optimise the ligand architecture for 89Zr-immunoPET imaging and develop a first-in-class regioselective dual chelator for use with the 89Zr/177Lu theranostic pair. 89Zr is used in a 4+ oxidation state, with a preference for octadentate coordination. The current ligand of choice for clinical application of 89Zr is the bacterially derived siderophore, desferrioxamine B (DFOB). DFOB forms a hexadentate coordinate complex with 89Zr, offering O,O coordination through three hydroxamic acid moieties. 89Zr(IV) is a hard acid and prefers coordination through hard base donor atoms, making hydroxamic acids an attractive choice as a coordinating moiety. It is believed that DFOB offers suboptimal coordination to 89Zr. It has been shown that by introducing a fourth hydroxamic acid to the DFOB scaffold, as seen in the ligand DFO*, that the stability of the metal-ligand complex can improve (90% in-tact complex after 24-hour challenge with 3000-fold excess of DFOB). Chain extended DFOB analogues can possess poor aqueous solubility, prompting further optimisation of the DFOB scaffold (DFOB-O3-PBH-O1, oxoDFO*, H3DFOSqOEt). In this work, an octadentate ligand DFO-8-WS-NCS was synthesised. DFO-8-WS-NCS included the addition of a fourth hydroxamic acid to allow octadentate coordination, a PEG4 unit which aimed to further improve aqueous solubility and a reactive functional group to allow mAb conjugation. Analysis of DFO-8-WS-NCS suggested greater aqueous solubility than DFO-NCS and demonstrated radiolabelling of 89Zr with high efficiency without significant mAb aggregation or degradation. Further biological studies are still required to assess the 89Zr-immunoPET imaging performance of [89Zr]Zr-DFO-8-WS-NCS-mAb and determine its biodistribution profile. Theranostic approaches can take two approaches. The first is to use ligand scaffolds that offer suboptimal coordination of one or both radiometals but present fewer biodistribution differences between imaging and therapy. The second is to use two separate ligand scaffolds that are optimised for each radiometal but can present greater differences in biodistribution across modalities. It was thought that the benefits provided by each approach could be achieved by designing a first-in-class dual chelator ligand that offered regioselective coordination of 89Zr and 177Lu. This ligand design utilised a DFOB chelate centre for 89Zr coordination, covalently linked to a DOTA chelate centre for 177Lu coordination. Literature suggested that under mild labelling conditions, DFOB would selectively coordinate 89Zr, while DOTA would selectively coordinate 177Lu. Metal-coordination studies on the discrete chelators, DFOB and DOTA produced data that correlated with literature, suggesting each chelator was selective for a single metal ion. A preliminary dual chelator, D2a, was synthesised by covalently linking DFOB to DOTA through an L-lysine linker. The L-lysine linker allowed for further functionalisation through the amine side chain. Further metal-coordination studies were conducted with D2a to confirm regioselectivity of the dual chelator scaffold. LC-MS and NMR (13C{1H}) spectroscopy analysis confirmed coordination of Zr(IV) by the DFOB chelate centre and coordination of Lu(III) by the DOTA chelate centre. The amine side chain of L-lysine was first functionalised with a reactive p-phenylene diisothiocyanate group to allow mAb conjugation, which showed undesirable intramolecular reactivity. D2-NCS was designed as a second-generation scaffold of D2a, with the installation of a PEG4 unit to the amine side chain of the L-lysine linker. This allowed functionalisation of the terminal amine of the PEG4 group with successful installation of p-phenylene diisothiocyanate. D2-NCS was synthesised in yields that facilitated radiolabelling and biological studies. D2-NCS was shown to effectively radiolabel 89Zr and 177Lu, with minimal impact to mAb integrity or degradation. Cell binding assays, biodistribution studies and 89Zr-immunoPET imaging suggested that D2-NCS had potential as a theranostic agent, displaying ideal cell internalisation characteristics for therapy, a comparable ex vivo biodistribution profile with both radiometals bound and similar 89Zr-immunoPET imaging performance to a matched control, DFO-NCS. In conclusion, this work demonstrated a new potential ligand for 89Zr-immunoPET that offers octadentate coordination to 89Zr with improved aqueous solubility over the current clinical standard, DFOB. This work also detailed the design of a first-in-class regioselective dual chelator for the 89Zr/177Lu theranostic pair. The dual chelator was shown to have potential as a theranostic agent, offering ideal diagnostic and therapeutic characteristics.