Figure 1.
– Sequence of DOM26h-196-61. Alignment of the primary sequence of the ASGPR specific dAb DOM26h-196-61 and the VHD2 isotype control sequence. DOM26h-196-61 residues identical to the VHD2 isotype control sequence are represented by ‘.’ Residue numbering was determined by the method of Kabat [34], with the residues contained within the three complementary determining regions (CDRs) underlined and in bold. In CDR3 of DOM26h-196-61, the symbol ‘∼’ has been used to facilitate alignment but does not represent a residue. B – Flow cytometry of liver targeting dAbs. ASGPR specific DOM26h-196-61 dAb (solid line) and VHD2 isotype control dAb (dotted line) were tested for binding to ASGPR positive primary human hepatocytes and the human hepatocarcinoma-derived cell line Huh7 by flow cytometry. Binding of DOM26h-196-61 to the ASGPR negative human cell line U937 is also shown for comparison. Detection of bound dAbs was demonstrated using a mouse monoclonal antibody specific for human VH domains and an Alexa 647 conjugated goat anti-mouse pAb. Staining of cells in the absence of dAb is also shown for comparison (shaded histograms).
Figure 2.
SDS-PAGE analysis of mIFNα2-dAb fusion protein purification.
Purification on protein A Streamline resin from clarified cell culture supernatant results in a single band of the expected molecular mass of approximately 33 KDa. CL = clarified supernatant, FT = flowthrough fraction, EL = eluted fraction.
Figure 3.
Surface plasmon resonance analysis of ASGPR specific dAbs and mIFNα2-dAb fusion proteins.
Murine ASGPR H1 antigen immobilised on CM5 chip surface was used to analyse binding kinetics of DOM26h-196-61 and mIFNα2-DOM26h-196-61 injected over the chip surface at a constant flow rate of 50 µl.min−1. mIFNα2-VHD2 isotype control was also injected over the chip surface as a negative control for antigen binding.
Figure 4.
In vitro activity of mIFNα2 formatted as dAb fusions.
Activity of the mIFNα2-dAb fusion proteins was tested in the B16-Blue™ assay and compared to unfused mIFNα2 standard. Error bars are not visible as they are smaller than the data points, but represent standard error of the mean of 3 independent experiments. mIFNα2-DOM26h-196-61 (dashed line, closed circles) and mIFNα2-VHD2 isotype control (dotted line, closed diamonds) showed comparable activity to the H6-mIFNα2 standard (solid line, closed squares), with only minor increases in the EC50.
Figure 5.
– Quantitative analysis of ASGPR dAb biodistribution. Quantitative analysis of 111In labelled dAb levels was carried out 3 hours after intravenous administration in BALB/c mice via tail vein injection of approximately 0.5 MBq radiolabelled dAb. Results show accumulation of radiolabeled ASGPR dAb DOM26h-196-61 in mouse liver is considerably higher than that observed with isotype control dAb. By contrast minimal uptake of either ASGPR dAb DOM26h-196-61 or VHD2 isotype control dAb was observed in any other organ besides kidney. Error bars shown represent standard deviation of the mean, n = 4. B– In vivo imaging of ASGPR specific dAbs. Localisation of 111In labelled dAbs in BALB/c mice at 3, 24 and 72 hours post injection. Images were captured following intravenous administration of 14–15 MBq of radiolabeled dAb via tail vein injection in BALB/c mice. Images show that signal is observed in kidney and bladder with both dAb molecules, whereas liver localisation is only observed with anti ASGPR VH dAb DOM26h-196-61.
Figure 6.
Quantitative analysis of mIFNα2 and mIFNα2-dAb biodistribution.
Quantitative analyses of 111In labelled mIFNα2 and fusion protein levels were carried out 3 hours after intravenous administration in BALB/c mice via tail vein injection of approximately 0.5 MBq radiolabeled compound. Results show accumulation of radiolabelled mIFNα2-dAb fusions in mouse liver is considerably higher than that observed with mIFNα2. Data also shows increased hepatic accumulation of mIFNα2-DOM26h-196-61 compared to mIFNα2-DOM26h-VHD2 isotype control. Error bars shown represent standard deviation of the mean, n = 4 (n = 3 in the case of mIFNα2).
Figure 7.
– In vivo imaging of mIFNα2 and mIFNα2-DOM26h-196-61. Localisation of 111In labelled mIFNα2 in beige SCID mouse and 111In labelled mIFNα2-DOM26h-196-61 in BALB/c mouse at 3, 24 and 72 hours post injection. Images were obtained following intravenous administration of 12–15 MBq of radiolabeled compound via tail vein injection in BALB/c mice. Images show that signal is observed in liver, kidney and bladder with IFN molecules. B– In vivo imaging of mIFNα2-VHD2 and mIFNα2-DOM26h-196-61. Localisation of 111In labelled mIFNα2-dAb fusions in BALB/c mice at 3, 24 and 72 hours post injection. Images were obtained following intravenous administration of 12–15 MBq of radiolabelled compound via tail vein injection in BALB/c mice. Images show that signal is observed in liver, kidney and bladder with both fusion proteins; however the extent of liver uptake is clearly higher in the animal injected with mIFNα2-DOM26h-196-61, in agreement with biodistribution data.