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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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Immunogenicity of rCrry-Ig. Rats were administered rCrry-Ig at days 0, 3, 7 and weekly thereafter, and blood was collected at certain time points. The presence of different antibodies against rCrry-Ig was detected by ELISA in the day 35 serum (a). Each bar represents a different animal. (b) Rabbit anti-Crry antiserum (shaded; positive control; mean fluorescence 399.6), buffer (solid line; mean fluorescence 51.3) or normal rabbit serum (dashed line; mean fluorescence 38.5) was incubated with CHO cells expressing rat Crry prior to incubation with active rat C. Deposition of C3b on the CHO cells was detected by flow cytometry. (c) Serum from an immune animal (shaded; mean fluorescence 41.72 ± 7.09), buffer (solid line; mean fluorescence 30.67) or normal rat serum (dashed line; mean fluorescence 38.5) was incubated with the CHO-Crry cells. The cells were attacked with C and C3b deposition detected as above. No increase in C3b deposition was observed following incubation with the immune sera. This histogram is representative of results from five different animals.
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fig5: Immunogenicity of rCrry-Ig. Rats were administered rCrry-Ig at days 0, 3, 7 and weekly thereafter, and blood was collected at certain time points. The presence of different antibodies against rCrry-Ig was detected by ELISA in the day 35 serum (a). Each bar represents a different animal. (b) Rabbit anti-Crry antiserum (shaded; positive control; mean fluorescence 399.6), buffer (solid line; mean fluorescence 51.3) or normal rabbit serum (dashed line; mean fluorescence 38.5) was incubated with CHO cells expressing rat Crry prior to incubation with active rat C. Deposition of C3b on the CHO cells was detected by flow cytometry. (c) Serum from an immune animal (shaded; mean fluorescence 41.72 ± 7.09), buffer (solid line; mean fluorescence 30.67) or normal rat serum (dashed line; mean fluorescence 38.5) was incubated with the CHO-Crry cells. The cells were attacked with C and C3b deposition detected as above. No increase in C3b deposition was observed following incubation with the immune sera. This histogram is representative of results from five different animals.

Mentions: We investigated whether generation of a Crry/antibody Fc chimeric protein created an immunogenic neoepitope at the join between the two proteins. Animals were administered rCrry-Ig intravenously on days 0, 3, 7 and weekly thereafter to 28 days. Blood was collected at weekly intervals, serum harvested and production of antibodies was assessed by ELISA. A weak immune response was detected to rCrry-Ig (Fig. 5a). All animals produced an IgM response in early bleeds (from day 7) with subsequent class switching to IgG1, IgG2b and IgG2c from day 21. The IgG2a response could not be assessed in this experiment as the ELISA coat contained the Fc of IgG2a resulting in cross-reactivity. Coating an ELISA with rCrry-Ig, sCrry and a control fusion protein with the same Fc revealed that the immune response had been mounted against all domains in the chimeric molecule and not just the join between Crry and the Fc domain (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)

Immunogenicity of rCrry-Ig. Rats were administered rCrry-Ig at days 0, 3, 7 and weekly thereafter, and blood was collected at certain time points. The presence of different antibodies against rCrry-Ig was detected by ELISA in the day 35 serum (a). Each bar represents a different animal. (b) Rabbit anti-Crry antiserum (shaded; positive control; mean fluorescence 399.6), buffer (solid line; mean fluorescence 51.3) or normal rabbit serum (dashed line; mean fluorescence 38.5) was incubated with CHO cells expressing rat Crry prior to incubation with active rat C. Deposition of C3b on the CHO cells was detected by flow cytometry. (c) Serum from an immune animal (shaded; mean fluorescence 41.72 ± 7.09), buffer (solid line; mean fluorescence 30.67) or normal rat serum (dashed line; mean fluorescence 38.5) was incubated with the CHO-Crry cells. The cells were attacked with C and C3b deposition detected as above. No increase in C3b deposition was observed following incubation with the immune sera. This histogram is representative of results from five different animals.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Immunogenicity of rCrry-Ig. Rats were administered rCrry-Ig at days 0, 3, 7 and weekly thereafter, and blood was collected at certain time points. The presence of different antibodies against rCrry-Ig was detected by ELISA in the day 35 serum (a). Each bar represents a different animal. (b) Rabbit anti-Crry antiserum (shaded; positive control; mean fluorescence 399.6), buffer (solid line; mean fluorescence 51.3) or normal rabbit serum (dashed line; mean fluorescence 38.5) was incubated with CHO cells expressing rat Crry prior to incubation with active rat C. Deposition of C3b on the CHO cells was detected by flow cytometry. (c) Serum from an immune animal (shaded; mean fluorescence 41.72 ± 7.09), buffer (solid line; mean fluorescence 30.67) or normal rat serum (dashed line; mean fluorescence 38.5) was incubated with the CHO-Crry cells. The cells were attacked with C and C3b deposition detected as above. No increase in C3b deposition was observed following incubation with the immune sera. This histogram is representative of results from five different animals.
Mentions: We investigated whether generation of a Crry/antibody Fc chimeric protein created an immunogenic neoepitope at the join between the two proteins. Animals were administered rCrry-Ig intravenously on days 0, 3, 7 and weekly thereafter to 28 days. Blood was collected at weekly intervals, serum harvested and production of antibodies was assessed by ELISA. A weak immune response was detected to rCrry-Ig (Fig. 5a). All animals produced an IgM response in early bleeds (from day 7) with subsequent class switching to IgG1, IgG2b and IgG2c from day 21. The IgG2a response could not be assessed in this experiment as the ELISA coat contained the Fc of IgG2a resulting in cross-reactivity. Coating an ELISA with rCrry-Ig, sCrry and a control fusion protein with the same Fc revealed that the immune response had been mounted against all domains in the chimeric molecule and not just the join between Crry and the Fc domain (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