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

McNeill E, Iqbal AJ, White GE, Patel J, Greaves DR, Channon KM - Sci Rep (2015)

Bottom Line: We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, British Heart Foundation Centre for Research Excellence, John Radcliffe Hospital , University of Oxford, U.K.

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.

No MeSH data available.


Related in: MedlinePlus

Pharmacokinetics of 35 K-Fc following ip injection.5 μg of 35 K-Fc was injected ip into C57bl/6J mice. The peritoneal cavity was lavaged with PBS and the plasma prepared from mice harvested over 7 days following injection (n = 3 per timepoint). The concentration of 35 K-Fc in peritoneal lavage, calculated as total remaining per animal (A), and plasma concentration (B) was determined by ELISA. The bioactivity of 35 K-Fc was determined using a chemotaxis bioassay. Plasma (10%) was used as a chemoattractant in a Boyden Chamber chemotaxis assay. The number of cells migrating towards the plasma samples was calculated as a migration index normalized to the amount of random migration seen to buffer alone (C). The concentration of RANTES in the plasma 5 was measured by ELISA (D). The plasma 35 K-Fc concentration was correlated with the plasma bioactivity (E). n = 3 per timepoint, Statistical analysis performed by one-way ANOVA and Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated. Linear correlation assessed by Spearman’s Test.
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f1: Pharmacokinetics of 35 K-Fc following ip injection.5 μg of 35 K-Fc was injected ip into C57bl/6J mice. The peritoneal cavity was lavaged with PBS and the plasma prepared from mice harvested over 7 days following injection (n = 3 per timepoint). The concentration of 35 K-Fc in peritoneal lavage, calculated as total remaining per animal (A), and plasma concentration (B) was determined by ELISA. The bioactivity of 35 K-Fc was determined using a chemotaxis bioassay. Plasma (10%) was used as a chemoattractant in a Boyden Chamber chemotaxis assay. The number of cells migrating towards the plasma samples was calculated as a migration index normalized to the amount of random migration seen to buffer alone (C). The concentration of RANTES in the plasma 5 was measured by ELISA (D). The plasma 35 K-Fc concentration was correlated with the plasma bioactivity (E). n = 3 per timepoint, Statistical analysis performed by one-way ANOVA and Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated. Linear correlation assessed by Spearman’s Test.

Mentions: Previous studies using our 35 K-Fc fusion protein had focused on in vitro or local efficacy in a peritonitis model and had not addressed the systemic availability of the protein13. 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 (harvested using either EDTA or Heparin anti-coagulants) (Supplementary Figure 1) to allow us to perform in vivo pharmacokinetic studies. We injected 5 ug 35 K-Fc ip into ApoE−/− mice and individual animals were sacrificed either pre-injection to provide a baseline or at intervals of 40 minutes to 7 days post-injection. The peritoneal cavity was lavaged to recover protein remaining locally and plasma prepared to assess systemic availability. 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 assay78. 35 K-Fc was taken up from the peritoneum over the first 6 hours, being completely gone by 48 hours (Fig. 1A). The level of 35 K-Fc remaining in the peritoneum significantly dropped at each tested time interval after the 40 min peak, until it was no longer present at 48 hours. A reciprocal appearance of the protein in the plasma was seen, with detectable 35 K-Fc already present 40 mins after injection (Fig. 1B). The plasma levels were maintained for at least 48 hours, after which time only small amounts were detectable. The peak plasma concentration was measured 2–6 hours following ip protein delivery, with plasma protein levels being significantly lower at the other timepoints sampled. This confirmed the systemic availability of the 35 K-Fc protein. When plasma from the injected animals was used as a chemoattractant in a Boyden Chamber chemotaxis assay, using murine Biogel-elicited macrophages, we detected a significant suppression of bioactivity within 2 hours of injection and lasting for 48 hours following a single injection of 35 K-Fc (Fig. 1C). This suppression of chemokine bioactivity occurred despite no change in plasma RANTES concentration, a primary CC chemokine found in ApoE−/− plasma (Fig. 1D). Although the suppression of chemokine bioactivity seen at earlier timepoints was not significant, the plasma bioactivity was significantly correlated with plasma 35 K-Fc concentration. A highly significant correlation was observed showing a direct relationship between plasma chemokine inhibitor and reduced chemokine bioactivity in the plasma (Fig. 1E).


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

McNeill E, Iqbal AJ, White GE, Patel J, Greaves DR, Channon KM - Sci Rep (2015)

Pharmacokinetics of 35 K-Fc following ip injection.5 μg of 35 K-Fc was injected ip into C57bl/6J mice. The peritoneal cavity was lavaged with PBS and the plasma prepared from mice harvested over 7 days following injection (n = 3 per timepoint). The concentration of 35 K-Fc in peritoneal lavage, calculated as total remaining per animal (A), and plasma concentration (B) was determined by ELISA. The bioactivity of 35 K-Fc was determined using a chemotaxis bioassay. Plasma (10%) was used as a chemoattractant in a Boyden Chamber chemotaxis assay. The number of cells migrating towards the plasma samples was calculated as a migration index normalized to the amount of random migration seen to buffer alone (C). The concentration of RANTES in the plasma 5 was measured by ELISA (D). The plasma 35 K-Fc concentration was correlated with the plasma bioactivity (E). n = 3 per timepoint, Statistical analysis performed by one-way ANOVA and Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated. Linear correlation assessed by Spearman’s Test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Pharmacokinetics of 35 K-Fc following ip injection.5 μg of 35 K-Fc was injected ip into C57bl/6J mice. The peritoneal cavity was lavaged with PBS and the plasma prepared from mice harvested over 7 days following injection (n = 3 per timepoint). The concentration of 35 K-Fc in peritoneal lavage, calculated as total remaining per animal (A), and plasma concentration (B) was determined by ELISA. The bioactivity of 35 K-Fc was determined using a chemotaxis bioassay. Plasma (10%) was used as a chemoattractant in a Boyden Chamber chemotaxis assay. The number of cells migrating towards the plasma samples was calculated as a migration index normalized to the amount of random migration seen to buffer alone (C). The concentration of RANTES in the plasma 5 was measured by ELISA (D). The plasma 35 K-Fc concentration was correlated with the plasma bioactivity (E). n = 3 per timepoint, Statistical analysis performed by one-way ANOVA and Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated. Linear correlation assessed by Spearman’s Test.
Mentions: Previous studies using our 35 K-Fc fusion protein had focused on in vitro or local efficacy in a peritonitis model and had not addressed the systemic availability of the protein13. 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 (harvested using either EDTA or Heparin anti-coagulants) (Supplementary Figure 1) to allow us to perform in vivo pharmacokinetic studies. We injected 5 ug 35 K-Fc ip into ApoE−/− mice and individual animals were sacrificed either pre-injection to provide a baseline or at intervals of 40 minutes to 7 days post-injection. The peritoneal cavity was lavaged to recover protein remaining locally and plasma prepared to assess systemic availability. 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 assay78. 35 K-Fc was taken up from the peritoneum over the first 6 hours, being completely gone by 48 hours (Fig. 1A). The level of 35 K-Fc remaining in the peritoneum significantly dropped at each tested time interval after the 40 min peak, until it was no longer present at 48 hours. A reciprocal appearance of the protein in the plasma was seen, with detectable 35 K-Fc already present 40 mins after injection (Fig. 1B). The plasma levels were maintained for at least 48 hours, after which time only small amounts were detectable. The peak plasma concentration was measured 2–6 hours following ip protein delivery, with plasma protein levels being significantly lower at the other timepoints sampled. This confirmed the systemic availability of the 35 K-Fc protein. When plasma from the injected animals was used as a chemoattractant in a Boyden Chamber chemotaxis assay, using murine Biogel-elicited macrophages, we detected a significant suppression of bioactivity within 2 hours of injection and lasting for 48 hours following a single injection of 35 K-Fc (Fig. 1C). This suppression of chemokine bioactivity occurred despite no change in plasma RANTES concentration, a primary CC chemokine found in ApoE−/− plasma (Fig. 1D). Although the suppression of chemokine bioactivity seen at earlier timepoints was not significant, the plasma bioactivity was significantly correlated with plasma 35 K-Fc concentration. A highly significant correlation was observed showing a direct relationship between plasma chemokine inhibitor and reduced chemokine bioactivity in the plasma (Fig. 1E).

Bottom Line: We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, British Heart Foundation Centre for Research Excellence, John Radcliffe Hospital , University of Oxford, U.K.

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.

No MeSH data available.


Related in: MedlinePlus