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Use of (64)Cu-labeled fibronectin domain with EGFR-overexpressing tumor xenograft: molecular imaging.

ABSTRACT: To assess the ability of an engineered epidermal growth factor receptor (EGFR)-binding fibronectin domain to serve as a positron emission tomographic (PET) probe for molecular imaging of EGFR in a xenograft mouse model.An EGFR-binding fibronectin domain (fibronectin abbreviated to Fn when bound) was site-specifically labeled with copper 64 ((64)Cu) (8 MBq/nmol). Copper 64-Fn binding was tested in cell cultures with varying EGFR expression. Stability in human and mouse serum was measured in vitro. Animal experiments were approved by the Stanford University Institutional Animal Care and Use Committee. Copper 64-Fn (approximately 2 MBq) was used for PET in mice (n = 5) bearing EGFR-overexpressing xenografted tumors (approximately 5-10 mm in diameter). Results of tomography were compared with those of ex vivo gamma counting of dissected tissues. Statistical analysis was performed with t tests and adjustment for multiple comparisons.Copper 64-Fn exhibited EGFR-dependent binding to multiple cell lines in culture. The tracer was stable for 24 hours in human and mouse serum at 37°C. The tracer exhibited good tumor localization (3.4% injected dose [ID]/g ± 1.0 [standard deviation] at 1 hour), retention (2.7% ID/g ± 0.6 at 24 hours), and specificity (8.6 ± 3.0 tumor-to-muscle ratio, 8.9 ± 4.7 tumor-to-blood ratio at 1 hour). Specific targeting was verified with low localization to low-expressing MDA-MB-435 tumors (0.7% ID/g ± 0.8 at 1 hour, P = .018); specificity was further demonstrated, as a nonbinding control fibronectin had low localization to EGFR-overexpressing xenografts (0.8% ID/g ± 0.2 at 1 hour, P = .013).The stability, low background, and target-specific tumor uptake and retention of the engineered fibronectin domain make it a promising EGFR molecular imaging agent. More broadly, it validates the fibronectin domain as a potential scaffold for a generation of various molecular imaging agents.

SUBMITTER: Hackel BJ

PROVIDER: S-EPMC3309798 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Use of (64)Cu-labeled fibronectin domain with EGFR-overexpressing tumor xenograft: molecular imaging.

Hackel Benjamin J BJ Kimura Richard H RH Gambhir Sanjiv S SS

Radiology 20120217 1

<h4>Purpose</h4>To assess the ability of an engineered epidermal growth factor receptor (EGFR)-binding fibronectin domain to serve as a positron emission tomographic (PET) probe for molecular imaging of EGFR in a xenograft mouse model.<h4>Materials and methods</h4>An EGFR-binding fibronectin domain (fibronectin abbreviated to Fn when bound) was site-specifically labeled with copper 64 ((64)Cu) (8 MBq/nmol). Copper 64-Fn binding was tested in cell cultures with varying EGFR expression. Stability ...[more]

PMID: 22344401

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Project description:The ability to detect and quantify macrophage accumulation can provide important diagnostic and prognostic information for atherosclerotic plaque. We have previously shown that LyP-1, a cyclic 9-amino acid peptide, binds to p32 proteins on activated macrophages, facilitating the visualization of atherosclerotic plaque with PET. Yet, the in vivo plaque accumulation of monomeric [(18)F]FBA-LyP-1 was low (0.31 ± 0.05%ID/g). To increase the avidity of LyP-1 constructs to p32, we synthesized a dendritic form of LyP-1 on solid phase using lysine as the core structural element. Imaging probes (FAM or 6-BAT) were conjugated to a lysine or cysteine on the dendrimer for optical and PET studies. The N-terminus of the dendrimer was further modified with an aminooxy group in order to conjugate LyP-1 and ARAL peptides bearing a ketone. Oxime ligation of peptides to both dendrimers resulted in (LyP-1)4- and (ARAL)4-dendrimers with optical (FAM) and PET probes (6-BAT). For PET-CT studies, (LyP-1)4- and (ARAL)4-dendrimer-6-BAT were labeled with (64)Cu (t1/2 = 12.7 h) and intravenously injected into the atherosclerotic (ApoE(-/-)) mice. After two hours of circulation, PET-CT coregistered images demonstrated greater uptake of the (LyP-1)4-dendrimer-(64)Cu than the (ARAL)4-dendrimer-(64)Cu in the aortic root and descending aorta. Ex vivo images and the biodistribution acquired at three hours after injection also demonstrated a significantly higher uptake of the (LyP-1)4-dendrimer-(64)Cu (1.1 ± 0.26%ID/g) than the (ARAL)4-dendrimer-(64)Cu (0.22 ± 0.05%ID/g) in the aorta. Similarly, subcutaneous injection of the LyP-1-dendrimeric carriers resulted in preferential accumulation in plaque-containing regions over 24 h. In the same model system, ex vivo fluorescence images within aortic plaque depict an increased accumulation and penetration of the (LyP-1)4-dendrimer-FAM as compared to the (ARAL)4-dendrimer-FAM. Taken together, the results suggest that the (LyP-1)4-dendrimer can be applied for in vivo PET imaging of plaque and that LyP-1 could be further exploited for the delivery of therapeutics with multivalent carriers or nanoparticles.

Project description:PURPOSE:The integrin ?v?6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for (64)Cu radiolabeling of A20FMDV2, an ?v?6 targeting peptide. PROCEDURES:Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4'-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with (64)Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted. RESULTS:All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37-50 °C for 15 min) with high specific activity (0.58-0.60 Ci/?mol). All radiotracers demonstrated ?v?6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers (64)Cu-CB-TE1A1P-1 and (64)Cu-BaBaSar-1 showed improved specificity for the ?v?6 positive tumor in vivo over (64)Cu-DOTA-1 and (64)Cu-NOTA-1 (+/- tumor uptake ratios-3.82?+/-?0.44, 3.82?±?0.41, 2.58?±?0.58, and 1.29?±?0.14, respectively). Of the four radiotracers, (64)Cu-NOTA-1 exhibited the highest liver uptake (10.83?±?0.1 % ID/g at 4 h). CONCLUSIONS:We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear "best candidate" for the (64)Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.

Dataset Information

Use of (64)Cu-labeled fibronectin domain with EGFR-overexpressing tumor xenograft: molecular imaging.

Publications

Use of (64)Cu-labeled fibronectin domain with EGFR-overexpressing tumor xenograft: molecular imaging.

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