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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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Inhibition of C by rCrry-Ig and sCrry. (a) rCrry-Ig was purified from cell culture supernatant by anti-Crry affinity chromatography and analysed by SDS-PAGE under non-reducing (7.5% gel; lane 1) or reducing conditions (10% gel; lane 2). (b) Antibody-sensitised erythrocytes were attacked with rat C and the ability of rCrry-Ig (■) and sCrry (▴) to inhibit CP-mediated haemolysis was compared with non-regulatory rat Ig (○). (c) Guinea pig erythrocytes were incubated with rat serum and the ability of rCrry-Ig (■), sCrry (▴) and rat Ig (○) to prevent AP-mediated haemolysis was determined. Each data point represents the mean value ±1S.D. of three determinations. (d) Methylamine inactivated C3 (C3MA) was incubated with fI in the presence of a cofactor: sCR1 (lane 3), rCrry-Ig (lane 4) or sCrry (lane 5). Ability of the reagent to act as cofactor was determined by SDS-PAGE on a 10% reduced gel followed by Western blotting. The blot was probed with anti-human C3c (cross-reactive with rat C3) followed by HRPO-conjugated anti-sheep Ig. Controls included C3MA only (lane 1) and C3MA with factor I (lane 2).
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fig3: Inhibition of C by rCrry-Ig and sCrry. (a) rCrry-Ig was purified from cell culture supernatant by anti-Crry affinity chromatography and analysed by SDS-PAGE under non-reducing (7.5% gel; lane 1) or reducing conditions (10% gel; lane 2). (b) Antibody-sensitised erythrocytes were attacked with rat C and the ability of rCrry-Ig (■) and sCrry (▴) to inhibit CP-mediated haemolysis was compared with non-regulatory rat Ig (○). (c) Guinea pig erythrocytes were incubated with rat serum and the ability of rCrry-Ig (■), sCrry (▴) and rat Ig (○) to prevent AP-mediated haemolysis was determined. Each data point represents the mean value ±1S.D. of three determinations. (d) Methylamine inactivated C3 (C3MA) was incubated with fI in the presence of a cofactor: sCR1 (lane 3), rCrry-Ig (lane 4) or sCrry (lane 5). Ability of the reagent to act as cofactor was determined by SDS-PAGE on a 10% reduced gel followed by Western blotting. The blot was probed with anti-human C3c (cross-reactive with rat C3) followed by HRPO-conjugated anti-sheep Ig. Controls included C3MA only (lane 1) and C3MA with factor I (lane 2).

Mentions: Given that the four N-terminal SCRs of rat Crry were identified as the minimal functional unit of Crry, a rCrry-Ig fusion protein was generated and purified as described for the sCrry reagents (Fig. 3a). The Fc domain was added to prolong the in vivo half-life as that of 4SCR was predicted to be short (Harris et al., 2002). The rCrry-Ig fusion protein had a molecular weight of 140 kDa by non-reducing SDS-PAGE and 70 kDa when the disulphide bonds at the hinge region were reduced (Fig. 3a; lane 2).


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)

Inhibition of C by rCrry-Ig and sCrry. (a) rCrry-Ig was purified from cell culture supernatant by anti-Crry affinity chromatography and analysed by SDS-PAGE under non-reducing (7.5% gel; lane 1) or reducing conditions (10% gel; lane 2). (b) Antibody-sensitised erythrocytes were attacked with rat C and the ability of rCrry-Ig (■) and sCrry (▴) to inhibit CP-mediated haemolysis was compared with non-regulatory rat Ig (○). (c) Guinea pig erythrocytes were incubated with rat serum and the ability of rCrry-Ig (■), sCrry (▴) and rat Ig (○) to prevent AP-mediated haemolysis was determined. Each data point represents the mean value ±1S.D. of three determinations. (d) Methylamine inactivated C3 (C3MA) was incubated with fI in the presence of a cofactor: sCR1 (lane 3), rCrry-Ig (lane 4) or sCrry (lane 5). Ability of the reagent to act as cofactor was determined by SDS-PAGE on a 10% reduced gel followed by Western blotting. The blot was probed with anti-human C3c (cross-reactive with rat C3) followed by HRPO-conjugated anti-sheep Ig. Controls included C3MA only (lane 1) and C3MA with factor I (lane 2).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Inhibition of C by rCrry-Ig and sCrry. (a) rCrry-Ig was purified from cell culture supernatant by anti-Crry affinity chromatography and analysed by SDS-PAGE under non-reducing (7.5% gel; lane 1) or reducing conditions (10% gel; lane 2). (b) Antibody-sensitised erythrocytes were attacked with rat C and the ability of rCrry-Ig (■) and sCrry (▴) to inhibit CP-mediated haemolysis was compared with non-regulatory rat Ig (○). (c) Guinea pig erythrocytes were incubated with rat serum and the ability of rCrry-Ig (■), sCrry (▴) and rat Ig (○) to prevent AP-mediated haemolysis was determined. Each data point represents the mean value ±1S.D. of three determinations. (d) Methylamine inactivated C3 (C3MA) was incubated with fI in the presence of a cofactor: sCR1 (lane 3), rCrry-Ig (lane 4) or sCrry (lane 5). Ability of the reagent to act as cofactor was determined by SDS-PAGE on a 10% reduced gel followed by Western blotting. The blot was probed with anti-human C3c (cross-reactive with rat C3) followed by HRPO-conjugated anti-sheep Ig. Controls included C3MA only (lane 1) and C3MA with factor I (lane 2).
Mentions: Given that the four N-terminal SCRs of rat Crry were identified as the minimal functional unit of Crry, a rCrry-Ig fusion protein was generated and purified as described for the sCrry reagents (Fig. 3a). The Fc domain was added to prolong the in vivo half-life as that of 4SCR was predicted to be short (Harris et al., 2002). The rCrry-Ig fusion protein had a molecular weight of 140 kDa by non-reducing SDS-PAGE and 70 kDa when the disulphide bonds at the hinge region were reduced (Fig. 3a; lane 2).

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