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Prevention of experimental autoimmune myasthenia gravis by rat Crry-Ig: A model agent for long-term complement inhibition in vivo.

Hepburn NJ, Chamberlain-Banoub JL, Williams AS, Morgan BP, Harris CL - Mol. Immunol. (2007)

Bottom Line: Fusion of these domains to rat IgG2a Fc generated an effective complement inhibitor (rCrry-Ig) with a circulating half-life prolonged from 7 min for Crry alone to 53 h for rCrry-Ig.Systemic administration of rCrry-Ig over 5 weeks generated a weak immune response to the recombinant agent, however this was predominantly IgM in nature and did not neutralise Crry function or cause clearance of the agent from plasma.The long half-life and low immunogenicity of this agent will be useful for therapy in chronic models of inflammatory disease in the rat.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom.

ABSTRACT
Despite its vital role in innate immunity, complement is involved in a number of inflammatory pathologies and has therefore become a therapeutic target. Most agents generated for anti-complement therapy have short half-lives in plasma, or have been of mouse or human origin, thereby limiting their use either to murine models of disease or to short-term therapy. Here we describe the generation of a long-acting rat therapeutic agent based on the rat complement inhibitor, Crry. Characterisation of various soluble forms of Crry demonstrated that the amino-terminal four short-consensus repeat domains were required for full regulatory and C3b-binding activities. Fusion of these domains to rat IgG2a Fc generated an effective complement inhibitor (rCrry-Ig) with a circulating half-life prolonged from 7 min for Crry alone to 53 h for rCrry-Ig. Systemic administration of rCrry-Ig over 5 weeks generated a weak immune response to the recombinant agent, however this was predominantly IgM in nature and did not neutralise Crry function or cause clearance of the agent from plasma. Administration of rCrry-Ig completely abrogated clinical disease in a rat model of myasthenia gravis whereas soluble Crry lacking the immunoglobulin Fc domain caused a partial response. rCrry-Ig not only ablated clinical disease, but also prevented C3 and C9 deposition at the neuromuscular junction and inhibited cellular infiltration at this site. The long half-life and low immunogenicity of this agent will be useful for therapy in chronic models of inflammatory disease in the rat.

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Effect of rCrry-Ig on rat C activity in vivo. Rats were administered rCrry-Ig (■) or sCrry (◊) and blood was collected at specific time intervals. The serum was isolated and used as a source of C to lyse antibody-coated erythrocytes. The lytic ability of the serum was compared to that of a pre-bleed. Each data point represents the mean of five animals ±1S.D.
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fig6: Effect of rCrry-Ig on rat C activity in vivo. Rats were administered rCrry-Ig (■) or sCrry (◊) and blood was collected at specific time intervals. The serum was isolated and used as a source of C to lyse antibody-coated erythrocytes. The lytic ability of the serum was compared to that of a pre-bleed. Each data point represents the mean of five animals ±1S.D.

Mentions: A single dose of the proteins was administered intravenously to normal rats in order to assess their C inhibitory ability. Serum was harvested at specific time intervals and haemolytic activity was assessed. Administration of sCrry had no discernable effect on serum haemolytic activity even at the early time points, likely due to rapid clearance, whereas animals administered rCrry-Ig had reduced serum haemolytic activity when compared to a pre-bleed, 40–50% for the first 8 h after administration (p < 0.05; Fig. 6). Reduction of serum haemolytic activity was detected for 24 h post-administration. It should be noted that this assay, because it involves dilution of test serum, underestimates the true inhibitory effect of the agent, which is highly concentration dependent.


Prevention of experimental autoimmune myasthenia gravis by rat Crry-Ig: A model agent for long-term complement inhibition in vivo.

Hepburn NJ, Chamberlain-Banoub JL, Williams AS, Morgan BP, Harris CL - Mol. Immunol. (2007)

Effect of rCrry-Ig on rat C activity in vivo. Rats were administered rCrry-Ig (■) or sCrry (◊) and blood was collected at specific time intervals. The serum was isolated and used as a source of C to lyse antibody-coated erythrocytes. The lytic ability of the serum was compared to that of a pre-bleed. Each data point represents the mean of five animals ±1S.D.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572221&req=5

fig6: Effect of rCrry-Ig on rat C activity in vivo. Rats were administered rCrry-Ig (■) or sCrry (◊) and blood was collected at specific time intervals. The serum was isolated and used as a source of C to lyse antibody-coated erythrocytes. The lytic ability of the serum was compared to that of a pre-bleed. Each data point represents the mean of five animals ±1S.D.
Mentions: A single dose of the proteins was administered intravenously to normal rats in order to assess their C inhibitory ability. Serum was harvested at specific time intervals and haemolytic activity was assessed. Administration of sCrry had no discernable effect on serum haemolytic activity even at the early time points, likely due to rapid clearance, whereas animals administered rCrry-Ig had reduced serum haemolytic activity when compared to a pre-bleed, 40–50% for the first 8 h after administration (p < 0.05; Fig. 6). Reduction of serum haemolytic activity was detected for 24 h post-administration. It should be noted that this assay, because it involves dilution of test serum, underestimates the true inhibitory effect of the agent, which is highly concentration dependent.

Bottom Line: Fusion of these domains to rat IgG2a Fc generated an effective complement inhibitor (rCrry-Ig) with a circulating half-life prolonged from 7 min for Crry alone to 53 h for rCrry-Ig.Systemic administration of rCrry-Ig over 5 weeks generated a weak immune response to the recombinant agent, however this was predominantly IgM in nature and did not neutralise Crry function or cause clearance of the agent from plasma.The long half-life and low immunogenicity of this agent will be useful for therapy in chronic models of inflammatory disease in the rat.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom.

ABSTRACT
Despite its vital role in innate immunity, complement is involved in a number of inflammatory pathologies and has therefore become a therapeutic target. Most agents generated for anti-complement therapy have short half-lives in plasma, or have been of mouse or human origin, thereby limiting their use either to murine models of disease or to short-term therapy. Here we describe the generation of a long-acting rat therapeutic agent based on the rat complement inhibitor, Crry. Characterisation of various soluble forms of Crry demonstrated that the amino-terminal four short-consensus repeat domains were required for full regulatory and C3b-binding activities. Fusion of these domains to rat IgG2a Fc generated an effective complement inhibitor (rCrry-Ig) with a circulating half-life prolonged from 7 min for Crry alone to 53 h for rCrry-Ig. Systemic administration of rCrry-Ig over 5 weeks generated a weak immune response to the recombinant agent, however this was predominantly IgM in nature and did not neutralise Crry function or cause clearance of the agent from plasma. Administration of rCrry-Ig completely abrogated clinical disease in a rat model of myasthenia gravis whereas soluble Crry lacking the immunoglobulin Fc domain caused a partial response. rCrry-Ig not only ablated clinical disease, but also prevented C3 and C9 deposition at the neuromuscular junction and inhibited cellular infiltration at this site. The long half-life and low immunogenicity of this agent will be useful for therapy in chronic models of inflammatory disease in the rat.

Show MeSH
Related in: MedlinePlus