*Article* **Thiol-Reactive PODS-Bearing Bifunctional Chelators for the Development of EGFR-Targeting [ 18F]AlF-A**ffi**body Conjugates**

**Chiara Da Pieve 1,\*, Ata Makarem 2, Stephen Turnock 3, Justyna Maczynska 3, Graham Smith <sup>1</sup> and Gabriela Kramer-Marek 3,\***


Academic Editors: Anne Roivainen and Xiang-Guo Li Received: 9 March 2020; Accepted: 27 March 2020; Published: 29 March 2020

**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.

**Keywords:** [ 18F]AlF; NOTA; NODAGA; PODS; thiol-reactive; linker; affibody molecule; bioconjugation; EGFR; tumor imaging

### **1. Introduction**

Reactive sulfhydryl groups of cysteine residues are attractive sites for the chemical attachment of dyes, chelators, or drugs to biomolecules. Targeting cysteine residues on biomolecules has several key benefits. Firstly, the presence of the thiol group, a highly reactive nucleophile, allows for a fast and selective reaction at physiological pH. The natural low abundance of accessible and reduced cysteine residues prevents the formation of heterogeneous mixtures of the bioconjugates. Moreover, a customized site-specific incorporation of cysteine residues into a biomolecule can be easily achieved [1]. Different classes of thiol-targeting electrophilic compounds have been used, with maleimides being the most common choice [2–4]. However, maleimide conjugates have shown instability mostly as a consequence

of thiol exchange reactions in vivo (e.g., retro-Michael addition) [5,6]. When maleimide-based bioconjugates are used as imaging agents, the above mentioned succinimidyl thioether linkage instability can lead to reduced accumulation in target tissues due to the release of the radioactive payload from the conjugate. Additionally, the thiol exchange reaction with endogenous thiol-containing biomolecules (e.g., albumin, cysteine and glutathione) can result in a higher background-to-noise ratio and a consequently reduced imaging contrast [7]. Thiol-reactive reagents have been investigated aiming at the formation of selective, fast, high-yielding and, most importantly, stable linkages with biomolecules [2,8–10]. Among the promising molecules, oxadiazolyl methyl sulfone-based compounds showed not only rapid and selective reaction with thiols in proteins but also the capacity of forming conjugates (via an oxadiazole–protein thiolate bond) which were more stable than those derived from maleimides [11,12]. The preparation of thiol-reactive bifunctional chelators and prosthetic groups containing the oxadiazolyl methylsulfone moiety (e.g., phenyloxadiazolyl methylsulfone or PODS) and their attachment to proteins and peptides have been examined [13–15]. Once radiolabeled, the conjugates were found to be more stable in vitro than the maleimide-derived counterparts. Moreover, when used in vivo, the oxadiazolyl methylsulfone linker-derived agents demonstrated reduced uptake in non-targeted tissues than the maleimide equivalents [13–15]. Based on these promising reports, we have prepared two novel phenyloxadiazolyl methylsulfone-containing (PODS) bifunctional chelators (NOTA-PODS and NODAGA-PODS) for the conjugation and aluminum fluoride ([18F]AlF) radiolabeling of a cysteine-containing biomolecule. For our study, an EGFR-targeting affibody molecule (ZEGFR:03115) was used as a thiol-bearing protein representative. The effect of the chelator structure on the synthesis and radiolabeling of the two conjugates ([18F]AlF-NOTA-PODS-ZEGFR:03115 and [ 18F]AlF-NODAGA-PODS-ZEGFR:03115) was studied together with their in vitro and in vivo profile. Additionally, a comparison was carried out with the maleimide-bearing [18F]AlF-NOTA-ZEGFR:03115 to benchmark in vitro and in vivo performance.
