Liver-Targeting of Interferon-Alpha with Tissue-Specific Domain Antibodies

PMCID: PMC3581439This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Pharmacokinetic Characteristics, Pharmacodynamic Effect and In Vivo Antiviral Efficacy of Liver-Targeted Interferon Alpha

Work carried out by JM was funded by the National Institutes of Health (NIH). NIH contract number is HHSN272201000039I/HHSN27200001/ A19

Analysis of IFN inducible gene expression in liver by mIFNα2-dAb fusions.

<p>Targeted mIFNα2 and non-targeted mIFNα2 were administered at 2μg/kg and 20μg/kg via intravenous injection. Vehicle control was also included. Levels of invariant and IFN inducible gene expression were analysed by TaqMan. Data shown are mean n = 4 animals. Error bars represent 95% CI.</p

<i>In vitro</i> activity of mIFNα2 formatted as dAb fusions.

<p>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-V_HD2 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.</p

Quantitative analysis of mIFNα2 and mIFNα2-dAb biodistribution.

<p>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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057263#s3" target="_blank">Results</a> 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-V_HD2 isotype control. Error bars shown represent standard deviation of the mean, n = 4 (n = 3 in the case of mIFNα2).</p

Surface plasmon resonance analysis of ASGPR specific dAbs and mIFNα2-dAb fusion proteins.

<p>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-V_HD2 isotype control was also injected over the chip surface as a negative control for antigen binding.</p

Pharmacokinetic analysis of ¹¹¹In-DOTA-mIFNα2-dAb fusions in kidney following intravenous administration.

<p>Targeted mIFNα2 (blue line) and non-targeted mIFNα2 isotype control dAb (red line) were administered at 20μg/kg via intravenous injection. Radioactivity levels were analysed in kidney by gamma counting. Data shown are mean n = 3 animals. Error bars represent s.e.m. Pharmacokinetic parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117847#pone.0117847.t005" target="_blank">Table 5</a>.</p

Pharmacokinetic analysis of ¹¹¹In-DOTA-mIFNα2-dAb fusions in liver following intravenous administration.

<p>Targeted mIFNα2 (blue line) and non-targeted mIFNα2 isotype control dAb (red line) were administered at 20μg/kg via intravenous injection. Radioactivity levels were analysed in liver by gamma counting. Data shown are mean n = 3 animals. Error bars represent s.e.m. Pharmacokinetic parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117847#pone.0117847.t004" target="_blank">Table 4</a>.</p

Pharmacokinetic analysis of mIFNα2-dAb fusions in serum following subcutaneous administration.

<p>Targeted mIFNα2 (red line) and non-targeted mIFNα2 isotype control dAb (grey line) were administered at 5mg/kg via subcutaneous injection. Compound levels were analysed in serum by mIFNα2 specific antibody capture and dAb specific detection. Data shown are mean n = 3 animals. Error bars represent s.e.m. Pharmacokinetic parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117847#pone.0117847.t002" target="_blank">Table 2</a>.</p