Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo

Eileen Mcneill,Jyoti Patel,David R Greaves,Keith M Channon,Asif J Iqbal,Gemma E White

doi:10.1038/srep17404

Abstract

Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

Highlights

Chronic inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, are a major cause of mortality and morbidity
We developed an ELISA using a combination of commercially available anti-35 K and anti-human IgG antibodies to detect 35 K-Fc fusion proteins, utilizing a high salt diluent to obtain a highly specific signal in mouse plasma (Supplementary Figure 1) to allow us to perform in vivo pharmacokinetic studies
ApoE−/− mice were used as we have previously shown that this hyperlipidemic model has elevated CC Chemokine levels in the plasma sufficient to allow the bioactivity of 35 K to be assessed in a chemotaxis assay[7,8]

Summary

Introduction

Chronic inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, are a major cause of mortality and morbidity. Binding of a molecule to an FcRn protects the molecule from lysosomal breakdown, instead the molecules are taken up via clathrin coated pits, whilst still bound to FcRn, which targets them to endosomal compartments allowing them to return to the cell-surface and return to circulation[11] This property has a dramatic effect on serum half life, with human IgG having a half-life of 23 days. Fc-Fusion proteins facilitate the purification of novel biologics, allowing standard antibody purification technologies to be utilized, and their detection for pharmacokinetic studies through highly specific Fc detection reagents[12] Whilst this can facilitate design and production of novel molecules under lab-scale conditions, rather than industrial, the ability to produce large protein amounts with minimal endotoxin contamination can still be expensive and time-consuming. Using hydrodynamic delivery we have demonstrated the in vivo utility of our CC-chemokine binding protein, mutant R89A 35 K-Fc, which we show for the first time is effective in an acute model of vascular inflammation

Methods

Results

Conclusion