FIGURE LEGENDS:
Figure 1. (A) Cartoon representations of the BsAb-1 and BsAb-2. BsAb-1 and BsAb-2 were constructed with scFv-2 and scFv-1, respectfully, covalently linked to the C-terminal end of the HC of the mAb-1 and mAb-2, respectfully, using Glycine-Serine linkers (shown in red). The BsAb molecules consist of a target binding orientation switch. The Fab region of BsAb-1 (consisting of mAb-1) binds to the same ligand as the scFv (scFv-1) component of BsAb-2; the Fab region of BsAb-2 (consisting of mAb-2) binds the same ligand as the scFv-2 region of BsAb-1. In the cartoon representation, the dark and light orange structures represent the antigen binding regions of the scFv-2 and Fab portions of mAb-2, whereas the dark and light black structures represent antigen binding regions of the scFv-1 and Fab portions of mAb-1. (B) The mean pharmacokinetic profiles of BsAb-1 and BsAb-2 following a single 5 mg/kg IV administration to cynomolgus monkeys. Data are the mean for three animals/timepoint for each molecule.
Figure 2: Mean concentrations (%ID/g) of (A)125I-labeled BsAb-1 and BsAb-2 and (B)111In- labeled BsAb-1 or BsAb-2 in male cynomolgus monkeys following a single IV administration of ~5 mg/kg (~0.015 mCi total 125I- and111In- labeled BsAb-1 or BsAb-2 mixed in equal amounts) in blood. Blood data are the mean (+/- standard deviation or SD) for N =3/timepoint for each molecule at 0.083, 1, 6, 24 and 48 hours post administration for each isotope; N=2/timepoint (+/- SD) for each molecule at 96 hours post administration for each isotope and N=1/timepoint for each molecule at 168 hours post administration for each isotope. The SD for the N=2 timepoint is displayed for illustrative purposes only. Statistical comparisons of the concentration versus time data were conducted between the two molecules labeled with the same isotope and between the same timepoint after administration. The ‘*’ symbol indicates statistically significant (P value <0.05) differences between BsAb-1 and BsAb-2 for the same isotope at the same timepoint post administration.
Figure 3: Percent of radioactive recovery data for (A) BsAb-1 and (B) BsAb-2 in male cynomolgus monkeys following a single IV administration of ~5 mg/kg (~0.015 mCi125I- and 111In- labeled BsAb-1 or BsAb-2 mixed in equal amounts) in the organs, urine, blood and carcass. Organs include liver, spleen, kidney, lung, skin, muscle and other tissue collected as per outlined in Materials and Methods section. Data are for N =1/timepoint for each isotope.
Figure 4: Immunofluorescence of BsAb-1, BsAb-2 and an ECD-based BsAb in cynomolgus monkey liver following a single IV administration of each molecule. In each panel green and red represent detection of the BsAb (using an anti-human IgG) and LSECs (via detection of the endothelial cell marker CD31/PECAM1), respectively. Immunofluorescence detection of (A) BsAb-1, (B) BsAb-2 and (C) ECD-based BsAb (used as a positive control from a previous study).13 Data are from representative liver sections from a single cynomolgus monkey. The scale bars represent 50 μm.
Figure 5: Mechanistic speculation of the intracellular trafficking pathways for BsAb-1 and BsAb-2 which lead to differential peripheral clearance.
Supplemental Figure 1: DSC thermograms of (A) BsAb-1 and (B) BsAb-2 in PBS, pH 7.2. All domains in BsAb-2 unfold at similar temperatures so the individual domain transitions are unresolved. The total ΔH for BsAb1 and BsAb2 are similar.