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.