Liver tissue distribution of the BsAbs in cynomolgus monkey using immunofluorescence
The biodistribution study revealed that BsAb-1 and BsAb-2 were distributed to the liver (Figure 4). Previous reports from our laboratories have shown BsAbs that have rapid peripheral clearance can show multifocal labeling of the sinusoidal lining around the hepatocytes consistent with endothelial cell association.10, 13 In an effort to delineate if the clearance difference between BsAb-1 and BsAb-2 were mechanistically a consequence of liver sinusoidal endothelial cells (LSEC), immunofluorescence detection of the BsAbs was evaluated at 6 hours following a single 5 mg/kg IV administration of each molecule on liver biopsies collected from the aforementioned cynomolgus monkey radiolabel biodistribution study. This timepoint was chosen based on the cynomolgus monkey pharmacokinetic profiles displaying a reasonable divergence in the peripheral exposure of the BsAbs (Figure 1B). Additional identification of LSECs was pursued by immunofluorescence detection of CD31 which are commonly used to identify endothelial cells in liver. Stored liver tissue sections from a previously reported ECD-based BsAb which had displayed liver deposition were included in the immunofluorescence staining as a positive control to provide a benchmark of LSEC association for interpretation of BsAb-1 and BsAb-2 data.13 At 6 hours post administration, neither BsAb-1 or BsAb-2 were detected in a distinguishable manner in the liver, but the positive control ECD-based BsAb was clearly detected (Figure 4). The data suggest that liver sinusoidal endothelial cells are not linked to the rapid clearance of BsAb-1 relative to BsAb-2.