Figure 1.
Schematic comparison of anti-CEA chimeric antibodies with PEGylated and non-PEGylated NIR dyes.
Four mPEG200 chains are covalently linked to each dye molecule, 4 of which are covalently linked to the chimeric anti-CEA antibody by amide bonds. The mPEG chains alter tissue biodistribution, allowing brighter liver metastases labeling and decreased accumulation in normal organs, particularly the liver.
Figure 2.
Whole body time sequence images of both PEGylated and non-PEGylated dye-anti-CEA antibody conjugates to evaluate total body biodistribution.
After the mice are sacrificed at the indicated time-points, they were opened up from sternum to pubis to completely expose the viscera. In 2a, DyLight 650 anti-CEA antibody conjugate is evaluated, with images taken at different time-points. The top row is the PEGylated dye anti-CEA antibody conjugate, while the bottom row is the non-PEGylated anti-CEA antibody conjugate. With the PEGylated anti-CEA antibody conjugate, there is minimal to no accumulation of dye in the viscera, while in the non-PEGylated anti-CEA antibody conjugate, there is accumulation of dye in the reticulo-endothelial system organs such as the liver and lung. Arrows in the bottom row point to lymph nodes that retained and were labeled by the dye. In 2b, DyLight 750 is evaluated, with images taken at different time-points. The top row is the PEGylated dye anti-CEA antibody conjugate, while the bottom row is the non-PEGylated anti-CEA antibody conguate. With the PEGylated anti-CEA antibody conjugate, there was minimal to no accumulation of dye in the viscera, while in the non-PEGylated anti-CEA antibody conjugate there was accumulation of dye in the liver and lung. Arrows in the bottom row point to lymph nodes that retained and were labeled by the dye.
Figure 3.
Liver tissue concentrations of the dye anti-CEA antibody conjugates over time.
The liver was assayed to demonstrate tissue accumulation of the non-PEGylated and PEGylated 650 and 750 dyes that were conjugated to chimeric anti-CEA antibody. The non-PEGylated 650 (panel A) and 750 dye antibody conjugates (panel B) accumulated to significantly higher levels in the liver compared to their respective PEGylated dye antibody conjugates.
Figure 4.
In 4a, the PEGylated 650 anti-CEA antibody conjugate was injected into the mouse 24 hours prior to imaging. While the liver metastases are partially visible on bright field imaging, the anti-CEA antibody conjugated to PEGylated dye accurately and selectively labeled the tumors showing the true extent of disease. The RFP signal visualizes less of the tumor in comparison to the 650 nm signal from the anti-CEA chimeric antibody conjugated to PEGylated dyes, as demonstrated by the arrowheads. Any deep tumor or tumor overlain by liver tissue was not visualized with the RFP signal due to the poor penetration and hemoglobin quenching of the 550 nm signal. Upper and lower panels show same mouse with liver lifted up in lower panel in order to show additional view of liver metastases. In 4b, the PEGylated 750 anti-CEA antibody conjugate was injected into the mouse 24 hours prior to imaging. The liver metastases in this case are barely visible on bright field imaging. The PEGylated anti-CEA antibody conjugate however, brightly labels the metastases showing the true extent of disease. The RFP signal did not demonstrate the true extent of disease due to low tissue penetration and hemoglobin quenching when compared to the 750 nm signal. Upper and lower panels show same mouse with liver lifted up in lower panel in order to show additional view of liver metastases.
Figure 5.
Demonstration of metastatic colon cancer cells adjacent to hepatic tissue.
H&E, confocal and fluorescence images of adjacent tissue samples are shown (Confocal and fluorescence images from same slide). The tumor margin was much better delineated when employing fluorescence microscopy.
Figure 6.
Specificity of anti-CEA antibody conjugated dyes as compared to free dyes.
In all images from A to D, the top panel of images is taken with the liver in anatomical position and the bottom panel is taken with the liver flipped upward to enable visualization of the undersurface. Image A is taken 24-anti-CEA antibody conjugate. Imaging at 750 nm clearly delineates all the HT-29-GFP liver metastases which are not completely visible on imaging with brightfield or GFP imaging (see arrows). Image B is taken 24 hours after injection of the 750 PEG dye only. No visible tumor labeling is noted, with little to no visualization of the dye throughout the animal, despite the GFP signal of the tumor being clearly detected. Image C is taken 24 hours after injection of the 650 PEG-anti-CEA antibody conjugate. There is very precise and clear labeling of tumor that is detected at 650 nm, which correlates well with the GFP signal. With the liver in the anatomical position, the gallbladder is also noted emerging from the underside of the liver. On the underside of the liver, a tumor is detected that is not visible on either GFP or brightfield imaging (see arrows). Image D is taken 24 hours after injection of the 650PEG free dye. There is non-specific distribution of the dye noted at 650 nm, with no selectivity for the tumor despite there being a robust GFP signal.