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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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Analysis of purified sCrry reagents by SDS-PAGE. The proteins were purified from cell culture supernatant by anti-Crry affinity chromatography and subjected to SDS-PAGE (11% gel). Lanes 1 and 3, sCrry non-reduced and reduced, respectively. Lanes 2 and 4, sCrry (3SCRs) non-reduced and reduced, respectively.
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fig1: Analysis of purified sCrry reagents by SDS-PAGE. The proteins were purified from cell culture supernatant by anti-Crry affinity chromatography and subjected to SDS-PAGE (11% gel). Lanes 1 and 3, sCrry non-reduced and reduced, respectively. Lanes 2 and 4, sCrry (3SCRs) non-reduced and reduced, respectively.

Mentions: To generate the soluble Crry proteins, DNA encoding the three or four N-terminal SCR domains of rat Crry was ligated into the pDR2ΔEF1α vector. CHO cells were transfected with the plasmids and stable cell lines were generated. Supernatant was harvested and proteins were purified using a monoclonal anti-Crry affinity column. Purified proteins were analysed by SDS-PAGE (Fig. 1). The molecular weight of sCrry was 28 kDa under reduced and non-reduced conditions. The molecular weight of sCrry (3SCRs) was 21 kDa under non-reducing conditions and 25 kDa under reducing conditions, an increase in weight characteristic of SCR-containing proteins. The molecular weights were corroborated by mass spectrometry (sCrry 28.248 Da; sCrry (3SCRs) 21.216 Da; 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)

Analysis of purified sCrry reagents by SDS-PAGE. The proteins were purified from cell culture supernatant by anti-Crry affinity chromatography and subjected to SDS-PAGE (11% gel). Lanes 1 and 3, sCrry non-reduced and reduced, respectively. Lanes 2 and 4, sCrry (3SCRs) non-reduced and reduced, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Analysis of purified sCrry reagents by SDS-PAGE. The proteins were purified from cell culture supernatant by anti-Crry affinity chromatography and subjected to SDS-PAGE (11% gel). Lanes 1 and 3, sCrry non-reduced and reduced, respectively. Lanes 2 and 4, sCrry (3SCRs) non-reduced and reduced, respectively.
Mentions: To generate the soluble Crry proteins, DNA encoding the three or four N-terminal SCR domains of rat Crry was ligated into the pDR2ΔEF1α vector. CHO cells were transfected with the plasmids and stable cell lines were generated. Supernatant was harvested and proteins were purified using a monoclonal anti-Crry affinity column. Purified proteins were analysed by SDS-PAGE (Fig. 1). The molecular weight of sCrry was 28 kDa under reduced and non-reduced conditions. The molecular weight of sCrry (3SCRs) was 21 kDa under non-reducing conditions and 25 kDa under reducing conditions, an increase in weight characteristic of SCR-containing proteins. The molecular weights were corroborated by mass spectrometry (sCrry 28.248 Da; sCrry (3SCRs) 21.216 Da; 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