Thiol-Reactive PODS-Bearing Bifunctional Chelators for the Development of EGFR-Targeting [18F]AlF-Affibody Conjugates

Abstract

Site-selective bioconjugation of cysteine-containing peptides and proteins is currently achieved via a maleimide–thiol reaction (Michael addition). When maleimide-functionalized chelators are used and the resulting bioconjugates are subsequently radiolabeled, instability has been observed both during radiosynthesis and post-injection in vivo, reducing radiochemical yield and negatively impacting performance. Recently, a phenyloxadiazolyl methylsulfone derivative (PODS) was proposed as an alternative to maleimide for the site-selective conjugation and radiolabeling of proteins, demonstrating improved in vitro stability and in vivo performance. Therefore, we have synthesized two novel PODS-bearing bifunctional chelators (NOTA-PODS and NODAGA-PODS) and attached them to the EGFR-targeting affibody molecule ZEGFR:03115. After radiolabeling with the aluminum fluoride complex ([18F]AlF), both conjugates showed good stability in murine serum. When injected in high EGFR-expressing tumor-bearing mice, [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 showed similar pharmacokinetics and a specific tumor uptake of 14.1 ± 5.3% and 16.7 ± 4.5% ID/g at 1 h post-injection, respectively. The current results are encouraging for using PODS as an alternative to maleimide-based thiol-selective bioconjugation reactions.

The aluminium-[18F]fluoride revolution: simple radiochemistry with a big impact for radiolabelled biomolecules

“…An EGFR targeted Affibody molecule [ 18 F]AlF-NOTA-Z EGFR:1907 has been developed and produced in a radiochemical yield of 15% (Su et al 2014 ). As all of these examples show, Affibody molecules are typically functionalised at their unique cystine residue via a maleimide-thiol Michael addition; however, instability has been observed during radiosynthesis and in vivo studies (Pieve et al 2020 ; Ponte et al 2016 ). Da Pieve et al ( 2020 ) have recently described phenyloxadiazolyl methylsulfone (PODS) derivatives of NOTA and NODAGA chelators which provides an alternative to maleimide chemistry and may overcome instability associated with the maleimide-thiol ligation at later time points.…”

“…As all of these examples show, Affibody molecules are typically functionalised at their unique cystine residue via a maleimide-thiol Michael addition; however, instability has been observed during radiosynthesis and in vivo studies (Pieve et al 2020 ; Ponte et al 2016 ). Da Pieve et al ( 2020 ) have recently described phenyloxadiazolyl methylsulfone (PODS) derivatives of NOTA and NODAGA chelators which provides an alternative to maleimide chemistry and may overcome instability associated with the maleimide-thiol ligation at later time points. [ 18 F]AlF-NOTA-PODS-Z EGFR:03115 and [ 18 F]AlF-NODAGA-PODS-Z EGFR:03115 radioconjugates were synthesised and showed specific tumour uptake 14.1 ± 5.3 and 16.7 ± 4.5%ID/g (1 h p.i.…”

“…[ 18 F]AlF-NOTA-PODS-Z EGFR:03115 and [ 18 F]AlF-NODAGA-PODS-Z EGFR:03115 radioconjugates were synthesised and showed specific tumour uptake 14.1 ± 5.3 and 16.7 ± 4.5%ID/g (1 h p.i. ), respectively (Pieve et al 2020 ).…”

Diagnosis of Glioblastoma by Immuno-Positron Emission Tomography

“…To date, several immuno-PET imaging tracers have been designed to target glioblastoma and have already proven successful in detecting gliomas in multiple preclinical models. These tracers target membrane proteins whose expression is altered in glioblastoma (including the EGFR, DLL4, EPHA2, CD47, AC133 antigen, and MT1-MMP) [ 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 106 , 107 ]: several components of the tumor microenvironment including vessels, macrophages, and extracellular matrix proteins [ 104 , 105 , 108 , 109 , 110 , 114 ]. Notably, [ 89 Zr]Zr-DFO-fresolimumab, an immuno-PET tracer based on a monoclonal antibody that can neutralize all mammalian isoforms of TGF-β was assayed in humans, penetrated recurrent high-grade gliomas but did not result in clinical benefit [ 109 ].…”

“…These tracers allow for evaluating multiple hallmarks [29] of gliomas and the treatment response in preclinical settings. Several immuno-PET tracers' [94][95][96][97][98][99][100][101]106,107] target membrane proteins whose expression is altered in glioblastoma including the Epidermal Growth Factor Receptor (EGFR), Delta-Like Ligand 4 (DLL4), Ephrin type-A receptor 2 (EPHA2), Cluster of differentiation 47 (CD47), the AC133 antigen, and the Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP14) (Figure 5). In vivo administration of these tracers showed high-specificcontrast imaging of the target in an MT1-MMP expressing glioblastoma tumor model and provided strong evidence for their utility as an alternative to non-specific imaging of glioblastoma.…”