Small-Animal PET Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with Site-Specific ¹⁸F-Labeled Protein Scaffold Molecules

Abstract

Human epidermal growth factor receptor type 2 (HER2) is a wellestablished tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of 18 F-labeled biomolecules for PET imaging of HER2 (HER2 PET ) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment. Methods: In this study, anti-HER2 monomeric and dimeric protein scaffold molecules [Z HER2:477 and (Z HER2:477 ) 2 , respectively] were radiofluorinated at a reasonable radiochemical yield (13%-18%) by use of site-specific oxime chemistry. The resulting radiofluorinated protein scaffold molecules were then evaluated as potential molecular probes for small-animal HER2 PET by use of a SKOV3 tumor-bearing mouse model. Results: The 4-18 F-fluorobenzaldehyde conjugated aminooxy-protein scaffolds [ 18 F-N-(4-fluorobenzylidene)oxime (FBO)-Z HER2:477 and 18 F-FBO-(Z HER2:477 ) 2 ] both displayed specific HER2-binding ability in vitro. Biodistribution and small-animal PET imaging studies further revealed that 18 F-FBO-Z HER2:477 showed rapid and high SKOV3 tumor accumulation and quick clearance from normal tissues, whereas 18 F-FBO-(Z HER2:477 ) 2 showed poor in vivo performance (low tumor uptake and tumorto-normal tissue ratios). The specificity of 18 F-FBO-Z HER2:477 for SKOV3 tumors was confirmed by its lower uptake on pretreatment of tumor-bearing mice with the HER2-targeting agents Z HER2 and trastuzumab. Moreover, small-animal PET imaging studies revealed that 18 F-FBO-Z HER2:477 produced higher-quality tumor imaging than 18 F-FBO-(Z HER2:477 ) 2 . 18 F-FBO-Z HER2:477 could clearly identify HER2-positive tumors with good contrast. Conclusion: Overall, these data demonstrate that 18 F-FBO-Z HER2:477 is a promising PET probe for imaging HER2 expression in living mice. It has a high potential for translation to clinical applications. The radiofluorination method developed can also be used as a general strategy for the site-specific labeling of other proteins with 18 F. The protein scaffold molecules used here are attractive for the further development of PET probes for other molecular targets.

Incorporation of a Triglutamyl Spacer Improves the Biodistribution of Synthetic Affibody Molecules Radiofluorinated at the N-Terminus via Oxime Formation with 18F-4-Fluorobenzaldehyde

“…Unlike Affibody molecules labeled using residualizing radiometal labels 22,23,32,35 , the renal retention of 18 F-N-(4-fluorobenzylidine)oxime-labeled Affibody molecules is quite low (Figure 9). This is in agreement with data showing rapid clearance of radioactivity from kidneys for other Affibody molecules labeled with 18 F-N-(4-fluorobenzylidine)oxime 19,29,30 . This is a strong indication that the Experiments in tumor-bearing mice confirmed that 18 F-FBO-E 3 -PEP4313 can target HER2-expressing xenografts with high specificity (Figure 8).…”

“…In fact, 18 F-fluorobenzaldehyde conjugation has been used for labeling of recombinant dimeric 19 and monomeric 29 anti-HER2 Affibody molecules (after conjugation of 2-…”

Phage display and molecular imaging: expanding fields of vision in living subjects

“…Chemical modifications with polyethylene glycol (PEG) or albumin binding peptides have been used to increase circulation time and enhance pharmacokinetic profiles (Chen et al 2004b). In addition, peptides with higher target binding affinity (Kimura et al 2009a) and multivalent target binding (Liu 2006) have been shown to elicit increased imaging signals compared to weaker binding and monomeric peptides; yet, in some cases monovalent proteins have been shown to exhibit better tumor uptake compared to higher affinity dimeric versions (Cheng et al 2008). The latter observation is interpreted to result from the effects of molecular size on tissue diffusion.…”

“…Initial clinical studies in human patients with these ErbB2-targeting affibody molecules, labeled with 111 In for SPECT imaging or 68 Ga for PET imaging, have also been reported (Baum et al 2006). In another study, oxime conjugation chemistry was used to prepare ErbB2-specific monomeric and dimeric affibody molecules labeled with 18 F for PET imaging (Cheng et al 2008). Unexpectedly, the monomer showed better in vivo performance despite the higher ErbB2 binding affinity of the dimer (Figure 3).…”

Noninvasive prediction of tumor responses to gemcitabine using positron emission tomography

“…Measurements of estrogen receptor expression by using [ 18 F]fluoroestradiol PET could be useful for stratifying breast cancer patients for estrogen-based therapy (9,10). PET using [4-18 F]fluorobenzaldehyde-conjugated aminooxy-protein scaffolds has been used in small animal models to detect tumoral Her2 expression (11). Similar approaches could guide the selection of chemotherapy regimes.…”