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A rheumatoid factor paradox: inhibition of rituximab effector function.

Jones JD, Shyu I, Newkirk MM, Rigby WF - Arthritis Res. Ther. (2013)

Bottom Line: Using human sera, addition of RTX resulted in rapid and profound (>50%) Daudi cell death that was complement dependent.Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF.This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Rituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC).

Methods: We developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated.

Results: Using human sera, addition of RTX resulted in rapid and profound (>50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis.

Conclusions: Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.

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RF inhibits rituximab effects by binding the Fc portion of RTX. A. Monoclonal IgM RF has an inhibitory effect on RTX-CDC when RTX (1 μg/ml) has an IgG1 heavy chain, but not when RTX has an IgG3 heavy chain (n = 3). B. Addition of RTX 10 μg/ml to PBMC in serum free media after addition of monoclonal IgM RF (n = 6) or IgA RF (n = 3) inhibits loss of CD19 by RTX-mediated trogocytosis. Inhibitory effects of RF found to be statistically significant (P < 0.05) are indicated by asterisks. Error bars represent mean standard error.
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Figure 7: RF inhibits rituximab effects by binding the Fc portion of RTX. A. Monoclonal IgM RF has an inhibitory effect on RTX-CDC when RTX (1 μg/ml) has an IgG1 heavy chain, but not when RTX has an IgG3 heavy chain (n = 3). B. Addition of RTX 10 μg/ml to PBMC in serum free media after addition of monoclonal IgM RF (n = 6) or IgA RF (n = 3) inhibits loss of CD19 by RTX-mediated trogocytosis. Inhibitory effects of RF found to be statistically significant (P < 0.05) are indicated by asterisks. Error bars represent mean standard error.

Mentions: IgM RF reacts with the cleft in the Cγ2/Cγ3 Fc portion of human IgG1, IgG2 and IgG4, but not IgG3 [7,8]. To prove the observed inhibitory effect of IgM RF requires a specific Fc domain, we compared RTX-CDC relative to IgG3 RTX. IgG3 RTX differs from RTX only in the presence of an IgG3 instead of IgG1 heavy chain. IgG3 RTX mediated RTX-CDC more efficiently than IgG1 RTX. More importantly, the addition of monoclonal IgM RF to RF- sera (n = 3) inhibited CDC with IgG1 RTX, but not with IgG3 RTX (Figure 7A).


A rheumatoid factor paradox: inhibition of rituximab effector function.

Jones JD, Shyu I, Newkirk MM, Rigby WF - Arthritis Res. Ther. (2013)

RF inhibits rituximab effects by binding the Fc portion of RTX. A. Monoclonal IgM RF has an inhibitory effect on RTX-CDC when RTX (1 μg/ml) has an IgG1 heavy chain, but not when RTX has an IgG3 heavy chain (n = 3). B. Addition of RTX 10 μg/ml to PBMC in serum free media after addition of monoclonal IgM RF (n = 6) or IgA RF (n = 3) inhibits loss of CD19 by RTX-mediated trogocytosis. Inhibitory effects of RF found to be statistically significant (P < 0.05) are indicated by asterisks. Error bars represent mean standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672827&req=5

Figure 7: RF inhibits rituximab effects by binding the Fc portion of RTX. A. Monoclonal IgM RF has an inhibitory effect on RTX-CDC when RTX (1 μg/ml) has an IgG1 heavy chain, but not when RTX has an IgG3 heavy chain (n = 3). B. Addition of RTX 10 μg/ml to PBMC in serum free media after addition of monoclonal IgM RF (n = 6) or IgA RF (n = 3) inhibits loss of CD19 by RTX-mediated trogocytosis. Inhibitory effects of RF found to be statistically significant (P < 0.05) are indicated by asterisks. Error bars represent mean standard error.
Mentions: IgM RF reacts with the cleft in the Cγ2/Cγ3 Fc portion of human IgG1, IgG2 and IgG4, but not IgG3 [7,8]. To prove the observed inhibitory effect of IgM RF requires a specific Fc domain, we compared RTX-CDC relative to IgG3 RTX. IgG3 RTX differs from RTX only in the presence of an IgG3 instead of IgG1 heavy chain. IgG3 RTX mediated RTX-CDC more efficiently than IgG1 RTX. More importantly, the addition of monoclonal IgM RF to RF- sera (n = 3) inhibited CDC with IgG1 RTX, but not with IgG3 RTX (Figure 7A).

Bottom Line: Using human sera, addition of RTX resulted in rapid and profound (>50%) Daudi cell death that was complement dependent.Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF.This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Rituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC).

Methods: We developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated.

Results: Using human sera, addition of RTX resulted in rapid and profound (>50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis.

Conclusions: Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.

Show MeSH
Related in: MedlinePlus