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.
Tc, for the potential use as radiopharmaceuticals for diagnostic imaging of breast cancer.
Methods:The conjugation was achieved in high yield using standard peptide coupling reactions between an amino modification on the aptamer and the activated carboxylic group on the ligands. The retention of the affinity of the MAG2 modified AptA for the MUC1 protein core was confirmed using a FID binding assay. The 2 labelled aptamers were separated from free 99m Tc using microcon filter separation and monitored by HPLC at all stages, to ensure that only radiolabelled aptamers were produced. The biodistribution properties of the two aptamer-radionuclide conjugates were analysed in MCF-7 tumour bearing mice and compared. Tc. The radiolabelled aptamers showed different tumour uptake and clearance, but will require further development prior to diagnostic use.
Results
Aptamers are characterized by a rapid renal clearance leading to a short in vivo circulating half-life. In order to use aptamers as anticancer therapeutic agents, their exposure time to the tumor has to be enhanced via increasing residency in the bloodstream. A way to achieve this goal is by conjugating the aptamer to poly(ethylene glycol) (PEG). Herein, we present the conjugation of a bifunctionalized anti-MUC1 aptamer (NH(2)-AptA-SR) with the (99m)Tc coordinating moiety MAG2 and either a conventional branched PEG or the comb-shaped PolyPEG via a two-step synthesis. The isolated products were radiolabeled with (99m)Tc and their biodistribution and tumor-targeting properties in MCF-7 tumor bearing mice were analyzed and compared.
Aptamers are known for their short in vivo circulating half-life and rapid renal clearance. Their conjugation to poly(ethylene glycol) (PEG) is a way to improve their residence in the body. Two aptamers (AptD and AptF), having a disulfide protected thiol modification on the 3' end, have been conjugated to maleimide activated PEGs of various molecular weights and structures (linear PEG20; branched PEG20 and 40; PolyPEG17, 40, and 60 kDa). The high yield coupling (70-80% in most of the cases) could be achieved using immobilized tris[2-carboxyethyl]phosphine hydrochloride (TCEP) as reducing agent at pH 4. The affinity of PEGylated AptD for its target was reduced by conjugation to linear PEG20 and branched PEG40, but not to branched PEG20 and PolyPEGs. This work demonstrates an alternative approach to PEGylation of aptamers, and that the effect of PEG on the affinity for the target varies according to the structure and conformation of the synthetic polymer.
The future of Zr-based immuno-PET is reliant upon the development of new chelators with improved stability compared to the currently used deferoxamine (DFO). Herein, we report the evaluation of the octadentate molecule DFO-HOPO (3) as a suitable chelator forZr and a more stable alternative to DFO. The molecule showed good potential for the future development of a DFO-HOPO-based bifunctional chelator (BFC) for the radiolabelling of biomolecules with Zr. This work broadens the selection of available chelators forZr in search of improved successors to DFO for clinical Zr-immuno-PET.
In head and neck squamous cell carcinoma (HNSCC), the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR mAb used for the treatment of HNSCC, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate non-invasive measurement of EGFR expression across the entire tumor burden and also allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. EGFR-specific Affibody molecule (ZEGFR:03115) was radiolabeled with zirconium-89 (Zr) and fluorine-18 (F). The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting Zr-DFO-ZEGFR:03115 (2.4-3.6 MBq, 2 µg) either together with or 30 mins after increasing amounts of unlabeled ZEGFR:03115 (1, 5, 10, 15 and 20 µg). PET images were acquired at 3, 24 and 48 h post-injection and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed andF-AlF-NOTA-ZEGFR:03115 (12 µg, 1.5-2 MBq/mouse) used to monitor receptor changes. In vivo studies demonstrated that co-injecting 10 µg of non-labeled molecules withZr-DFO-ZEGFR:03115 allows for clear tumor visualization 3 h post-injection. The radioconjugate tumor accumulation was EGFR-specific and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration and receptor expression in tumor tissues. Additionally, F-AlF-NOTA-ZEGFR:03115 could measure receptor downregulation in response to EGFR inhibition. ZEGFR03115-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.
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