18F Site-Specific Labelling of a Single-Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography

Figures

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  Figure 1. Flow cytometry analysis comparing the binding of non-labelled targeted-ScFv and [18F]FBEM radiolabelled targeted-ScFv. The purple filled curve represents antibody binding to non-activated platelets, and the green curve shows binding to activated platelets. (A) shows that scFvs’ ability to bind to activated platelets is not altered by the [18F]FBEM radiolabelling process. (B) shows that the mutated-ScFv does not bind to activated platelets. Representative results are shown.

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  Figure 2. Biodistribution of [18F]FBEM (A) radiolabelled targeted-ScFv and (B) mutated-ScFv. Radioactivity uptake in the different organs was measured using a gamma counter and expressed as a percentage of injected dose per gram of tissue. The injured vessel (left carotid artery) only shows significant uptake of [18F]FBEM-targeted-ScFv at the 15 min time point (p < 0.009). Clearance from the blood, and rapid elimination via the kidney, is demonstrated by the high radioactivity uptake in the kidneys in earlier time points and the increasing radioactivity present in the urine at later time points. Skeletal muscle had a high uptake for both [18F]FBEM-labelled targeted antibody targeted-ScFv and mutated-ScFv (n = 5–6). Data were subjected to paired two-tailed Student t-test (** indicates p ≤ 0.01).

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  Figure 3. A comparison of radioactivity uptake between injured and intact vessels. (A) represents radioactivity uptake in mice that were injected with [18F]FBEM-targeted-ScFv over time. At the 15-min time point, there was a significant difference between the injured vessel (left carotid) and the intact vessel (right carotid). (B) shows that there was no significant difference in radioactivity uptake, between the injured and non-injured vessels, at all time points investigated (n = 5). (C,D) show the same comparison for the S[18F]FB-labelled scFv, with significant uptake of S[18F]FB-targeted ScFv observed at 5 min p.i. For both tracers, the values have been normalised to having brain tissue as the background tissue. Data were subjected to 2-tailed student t-test (** indicates p ≤ 0.01).

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  Figure 4. Co-registered PET/MRI scans of a mouse injected with 18F-scFv-cys (A) shows co-registered PET/MRI scans of a mouse injected with [18F]FBEM-radiolabelled targeted-ScFv. Both views show uptake in the target area (white arrow) where the FeCl3 injury was performed in transverse and coronal views. (B) shows a co-registered PET/MRI image of a mouse injected with [18F]FBEM-radiolabelled mutated-ScFv, and there is minimal radioactivity uptake in the target area. Representative results are shown.

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  Figure 5. Results from image analysis of ROIs. The difference in radioactivity uptake between the injured vessel and intact vessel is plotted against scan time. The red line represents the difference in vessel uptake in mice injected with 18F-targeted-ScFv, whilst the blue line represents the difference in vessel uptake in mice injected with 18F-mutated-ScFv. For mice injected with [18F]FBEM-labelled targeted-ScFv, the difference in radioactive uptake, between injured and non-injured, is only observed at later time points after 45 min into scan time (n = 5). Means were compared by use of a 1-way ANOVA.