Efficient [¹⁸F]AlF Radiolabeling of Z_HER3:8698 Affibody Molecule for Imaging of HER3 Positive Tumors

Abstract

The human epidermal growth factor receptor 3 (HER3) is overexpressed in several cancers, being linked to a more resistant phenotype and hence leading to poor patient prognosis. Imaging HER3 is challenging owing to the modest receptor number (<50000 receptors/cell) in overexpressing cancer cells. Therefore, to image HER3 in vivo, high target affinity PET probes need to be developed. This work describes two different [(18)F]AlF radiolabeling strategies of the ZHER3:8698 affibody molecule specifically targeting HER3. The one-pot radiolabeling of ZHER3:8698 performed at 100 °C and using 1,4,7-triazanonane-1,4,7-triacetate (NOTA) as chelator resulted in radiolabeled products with variable purity attributed to radioconjugate thermolysis. An alternative approach based on the inverse electron demand Diels-Alder (IEDDA) reaction between a novel tetrazine functionalized 1,4,7-triazacyclononane-1,4-diacetate (NODA) chelator and the trans-cyclooctene (TCO) functionalized affibody molecule was also investigated. This method enabled the radiolabeling of the protein at room temperature. The [(18)F]AlF-NOTA-ZHER3:8698 and [(18)F]AlF-NODA-ZHER3:8698 conjugates showed a specific uptake at 1 h after injection in high HER3-expressing MCF-7 tumors of 4.36 ± 0.92% ID/g and 4.96 ± 0.65% ID/g, respectively. The current results are encouraging for further investigation of [(18)F]AlF-NOTA-ZHER3:8698 as a HER3 imaging agent.

A review of approaches to 18F radiolabelling affinity peptides and proteins

“…A reaction by‐product was observed, presumed to be an APP degradation product as a result of the radiolabelling conditions; as a result, Da Pieve et al report the requirement for both RP‐HPLC and SPE purification to ensure a final RCP of >98%. In vitro analysis using HER3 expressing cells confirmed retention of [ 18 F]Al‐F‐NOTA‐affibody target specificity and affinity, indicated by preservation of the affibody's low nanomolar binding constant . These results were echoed by the in vivo analysis, using HER3 tumour‐bearing mice, where successful tumour targeting and fast blood clearance of Al[ 18 F]NOTA‐affibody were observed …”

“…Da Pieve et al used the ([ 18 F]AlF) 2+ method to radiolabel a HER3‐specific affibody with a view to assessing its usefulness as radiotracer in oncology; the method mirrored conditions reported by Glaser et al The affibody was derivatised with 1,4,7‐triazacyclononane‐1,4,7‐triacetic acid (NOTA) and incubated with AlCl 3 . Final incubation of the complex with 18 F gave rise to yields of 38.8 ± 5.8% non‐decay‐corrected (n.d.c) and molar activity measurements of 6.0 to 11.9 GBq/μmol were reported .…”

“…Da Pieve et al used the ([ 18 F]AlF) 2+ method to radiolabel a HER3‐specific affibody with a view to assessing its usefulness as radiotracer in oncology; the method mirrored conditions reported by Glaser et al The affibody was derivatised with 1,4,7‐triazacyclononane‐1,4,7‐triacetic acid (NOTA) and incubated with AlCl 3 . Final incubation of the complex with 18 F gave rise to yields of 38.8 ± 5.8% non‐decay‐corrected (n.d.c) and molar activity measurements of 6.0 to 11.9 GBq/μmol were reported . A reaction by‐product was observed, presumed to be an APP degradation product as a result of the radiolabelling conditions; as a result, Da Pieve et al report the requirement for both RP‐HPLC and SPE purification to ensure a final RCP of >98%.…”

“…Final incubation of the complex with 18 F gave rise to yields of 38.8 ± 5.8% non‐decay‐corrected (n.d.c) and molar activity measurements of 6.0 to 11.9 GBq/μmol were reported . A reaction by‐product was observed, presumed to be an APP degradation product as a result of the radiolabelling conditions; as a result, Da Pieve et al report the requirement for both RP‐HPLC and SPE purification to ensure a final RCP of >98%. In vitro analysis using HER3 expressing cells confirmed retention of [ 18 F]Al‐F‐NOTA‐affibody target specificity and affinity, indicated by preservation of the affibody's low nanomolar binding constant .…”

18F‐Labeling of Sensitive Biomolecules for Positron Emission Tomography

“…It is worth noting that structural rearrangement of adduct dihydropyridazine and aromatization, as identified by chromatography, could occur during IEDDA reactions. [137, 139-140] …”