Summary                         

Atherosclerosis is characterized by gradual plaque build-up in the middle and large arteries and is the major cause of cardiovascular disease. Determining which plaques are prone to rupture and cause potential lethal effects (e.g., myocardial infarction) could greatly reduce the potential bad outcomes.

CD40 is a co-stimulatory molecule present in various cells in the plaque and has been shown to correlate with plaque vulnerability. In the present experiment, a murine monoclonal antibody (mAb) against CD40 (a costimulatory molecule directly linked to plaque progression and present on many cells within the lesion) was labeled with Zirconium-89 to test its applicability to detect lesions in a mouse model of atherosclerosis using nanoScanPET/CT. Results show that it can successfully detect CD40 in atherosclerotic lesions. In wild type mice without plaques, no signal was found. In conclusion, CD40 is a potential marker for PET imaging of plaque inflammation and vulnerability.

Results from nanoScan PET/CT

8 weeks old ApoE-/- mice were fed a high fat diet for 14 weeks were intravenously injected with either ⁸⁹Zr-anti-CD40 mAb (100µg, 5MBq in 150µl, n=6, “low dose”) or ⁸⁹Zr-anti-CD40 mAb supplemented with additional cold mAb (1000µg, 5MBq in 200µl, n=6, “ high dose”). PET scans followed by a contrast enhanced CT scan to visualize the blood vasculature were performed at day 3 and 7 p.i. At day 7, immediately after PET imaging, animals were sacrificed and perfused to remove the blood content. The aorta and its main branch points along with all other organs were carefully
removed for assessment of ex vivo biodistribution and further analysis.

A computed tomography (CT) (5min) was performed to acquire morphological data for image processing, reconstruction and identification of organs and tissues of interest. The CT was followed by a 60min PET scan. At the end of each PET scan an additional CT scan (2min) was acquired while injecting contrast agent (Iomeron 400). Contrast agent was injected intravenously via tail vein catheter (250µl, speed rate of 200µl/min) using an intravenous pump, to visualize the cardiovascular structures. PET image reconstruction was performed using a fully 3-dimensional reconstruction algorithm (Tera-TomoTM, Mediso Ltd., Budapest, Hungary) with 4 iterations and 6 subsets, and an isotropic 0.4mm voxel dimension. Scattering and attenuation correction were applied.

Regions of interest (ROIs) were drawn on the aorta arch, spleen, liver, muscle, and lymph nodes and tracer concentration were extracted from the PET images. Blood tracer content was determined by drawing a ROI on the left ventricle (LV). Radioactivity values were decay corrected to the injection time, and the radioactivity uptake was calculated as the percentage of the injected dose per gram of tissue (%ID/g). The aorta-to-blood ratio was calculated by dividing the %ID/g of the aorta arch with the %ID/g of total blood determined from the LV. The ex vivo biodistribution was assessed with gamma counter at 7 days post injection after the last PET/CT scan.

Results show:

• In “low dose” mice, significantly higher aorta-to-blood ratios in ApoE−/− mice compared to WT animals at day 3 and 7 p.i.
• High uptake was also observed in the spleen and lymph node of ApoE-/- mice at day 3 and 7 p.i., which is in accordance with the high level of CD40 expression in these organs. Liver uptake was comparable between WT and ApoE-/- mice

High expression of CD40 in healthy organs might induce a sink effect that alters the antibody kinetics and atherosclerotic plaque targeting. In certain cases, a saturation of the sink organs can improve targeting to the organs of interest. To investigate this, the effect of a higher mass dose (“high dose” of 1000µg) on the pharmacokinetics and aorta uptake was evaluated:

• Higher uptake was observed in the aorta and in the blood compared to “low dose” at day 3 and 7 p.i. indicating CD40 sink effect
• Aorta-to-blood ratio was lower on both days compared to “low dose”

→ This indicates that a high mass dose of ⁸⁹Zr-anti-CD40 mAb is not favorable for PET imaging of plaques due to the slower blood kinetics that leads to poor image contrast. In the CD40-rich organs, spleen, and lymph nodes, we observed lower uptake of ⁸⁹Zr-anti-CD40 mAb in the “high dose” compared to the “low dose”, indicating a blocking effect in these organs and demonstrating the specificity of the tracer for targeting CD40 in vivo.

To validate the PET imaging observations and results, we performed ex vivo biodistribution immediately after PET imaging acquisition at day 7. Blood samples were also collected at different time points to assess the blood kinetics of ⁸⁹Zr-anti-CD40 mAb in WT and ApoE−/− mice at “low dose” and “high dose”. As the aortas were too small for accurate quantification of ex vivo biodistribution, aortas were used for autoradiography analysis.

• Ex vivo biodistribution results showed high uptake of ⁸⁹Zr-anti-CD40 mAb “low dose” in spleen and lymph nodes at day 7 p.i., which is in accordance with the PET imaging quantification
• Uptake of the tracer in spleen and lymph nodes was significantly lower when the mice were dosed with the “high dose” tracer (1000µg) compared to the “low dose” tracer (100µg)
• At day 3 and 7 a faster washout of the tracer from the blood was observed in the ApoE−/− compared the to the WT mice; slower kinetics were observed when mice were injected with the “high dose”
• At day 7 p.i., blood levels were significant higher in the ApoE−/− “high dose” group compared to the ApoE−/− “low dose” group
• Autoradiography images showed accumulation of the ⁸⁹Zr-anti-CD40 mAb in atherosclerotic plaques of the aorta and the uptake correlated well with the location of plaques in these tissues as identified by Oil red O staining. No plaques were observed in the WT mice. Quantification of autoradiography signals showed a significantly higher uptake in the plaques of the ApoE-/- “low dose” group compared to the ApoE-/- “high dose” group.

→ These results are in accordance with the PET imaging quantifications.

Full article on mdpi.com