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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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SPR analysis of the interaction of the sCrry reagents with rat C3b. Rat C3b was coupled covalently to the chip surface using the internal thioester bond and the interaction with sCrry (a) and sCrry (3SCRs) (b) was analysed. The affinity of the interaction was analysed by steady state kinetics (see inset).
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fig2: SPR analysis of the interaction of the sCrry reagents with rat C3b. Rat C3b was coupled covalently to the chip surface using the internal thioester bond and the interaction with sCrry (a) and sCrry (3SCRs) (b) was analysed. The affinity of the interaction was analysed by steady state kinetics (see inset).

Mentions: To identify the minimal functional unit of Crry, the binding of sCrry and sCrry (3SCRs) to rat C3b was compared using SPR. Rat C3b was coupled to the chip surface and different concentrations of each protein flowed across the surface. The binding of sCrry (3SCRs) to C3b was dramatically reduced compared to sCrry (Fig. 2a and b). The affinity of the two proteins for C3b was calculated using steady state analysis (insets Fig. 2a and b). The dissociation equilibrium constant (KD) for sCrry protein interaction with C3b was 5.1 × 10−6 M (χ2 = 0.4) whereas that for the sCrry (3SCRs) protein was too low to calculate accurately; there was minimal binding even at 33 μM. The sCrry (3SCRs) protein also demonstrated weak inhibitory activity in AP haemolysis assays (IH50 = 545 nM ± 51; sCrry IH50 = 74.1 nM ± 1.8; data not shown).


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)

SPR analysis of the interaction of the sCrry reagents with rat C3b. Rat C3b was coupled covalently to the chip surface using the internal thioester bond and the interaction with sCrry (a) and sCrry (3SCRs) (b) was analysed. The affinity of the interaction was analysed by steady state kinetics (see inset).
© Copyright Policy
Related In: Results  -  Collection

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

fig2: SPR analysis of the interaction of the sCrry reagents with rat C3b. Rat C3b was coupled covalently to the chip surface using the internal thioester bond and the interaction with sCrry (a) and sCrry (3SCRs) (b) was analysed. The affinity of the interaction was analysed by steady state kinetics (see inset).
Mentions: To identify the minimal functional unit of Crry, the binding of sCrry and sCrry (3SCRs) to rat C3b was compared using SPR. Rat C3b was coupled to the chip surface and different concentrations of each protein flowed across the surface. The binding of sCrry (3SCRs) to C3b was dramatically reduced compared to sCrry (Fig. 2a and b). The affinity of the two proteins for C3b was calculated using steady state analysis (insets Fig. 2a and b). The dissociation equilibrium constant (KD) for sCrry protein interaction with C3b was 5.1 × 10−6 M (χ2 = 0.4) whereas that for the sCrry (3SCRs) protein was too low to calculate accurately; there was minimal binding even at 33 μM. The sCrry (3SCRs) protein also demonstrated weak inhibitory activity in AP haemolysis assays (IH50 = 545 nM ± 51; sCrry IH50 = 74.1 nM ± 1.8; data not shown).

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