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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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IgM rheumatoid factor does not prevent RTX attachment to CD20. A. RF inhibits B cell death whether RTX is first added to cells followed later by RF addition (Stepwise), or if RTX and RF are added simultaneously (n = 3). Error bars represent mean standard error. B. Representative data (n = 2) showing that RTX blocks binding of anti-human CD20 APC-Cy7, and addition of RF to RTX does not lessen this blocking outcome.
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Figure 6: IgM rheumatoid factor does not prevent RTX attachment to CD20. A. RF inhibits B cell death whether RTX is first added to cells followed later by RF addition (Stepwise), or if RTX and RF are added simultaneously (n = 3). Error bars represent mean standard error. B. Representative data (n = 2) showing that RTX blocks binding of anti-human CD20 APC-Cy7, and addition of RF to RTX does not lessen this blocking outcome.

Mentions: The effect of RF on RTX-CDC (and C1q deposition) might be due to its capture of RTX in fluid phase, thus preventing binding to CD20 on the B cell. Alternatively, RF engagement of the Fc domain of RTX might block the recruitment of complement after RTX had engaged with CD20. To investigate these possibilities, we compared the resultant CDC from preincubation (15 minutes) of Daudi cells with RTX (to ensure B cell binding) followed by addition of IgM RF and serum relative to the simultaneous addition of RTX, RF and serum (Figure 6A). RF blocked RTX-CDC independent of the time of addition (stepwise vs. simultaneous) (n = 3, P = 0.34). These data suggest that RF inhibits complement binding to the Fc portion of RTX after it has bound CD20. This interpretation was supported by the observation that RF had no effect on RTX binding to CD20, as measured by the lack of modulation of the ability of RTX to block the binding of fluorochrome-labeled anti-human CD20 (APC-Cy7) to Daudi cells (Figure 6B). Thus, RF does not limit the ability of RTX to bind CD20 on the B cell. These data suggest that RF inhibits C3b deposition and cytotoxicity by preventing C1q binding to the Fc portion of RTX.


A rheumatoid factor paradox: inhibition of rituximab effector function.

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

IgM rheumatoid factor does not prevent RTX attachment to CD20. A. RF inhibits B cell death whether RTX is first added to cells followed later by RF addition (Stepwise), or if RTX and RF are added simultaneously (n = 3). Error bars represent mean standard error. B. Representative data (n = 2) showing that RTX blocks binding of anti-human CD20 APC-Cy7, and addition of RF to RTX does not lessen this blocking outcome.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: IgM rheumatoid factor does not prevent RTX attachment to CD20. A. RF inhibits B cell death whether RTX is first added to cells followed later by RF addition (Stepwise), or if RTX and RF are added simultaneously (n = 3). Error bars represent mean standard error. B. Representative data (n = 2) showing that RTX blocks binding of anti-human CD20 APC-Cy7, and addition of RF to RTX does not lessen this blocking outcome.
Mentions: The effect of RF on RTX-CDC (and C1q deposition) might be due to its capture of RTX in fluid phase, thus preventing binding to CD20 on the B cell. Alternatively, RF engagement of the Fc domain of RTX might block the recruitment of complement after RTX had engaged with CD20. To investigate these possibilities, we compared the resultant CDC from preincubation (15 minutes) of Daudi cells with RTX (to ensure B cell binding) followed by addition of IgM RF and serum relative to the simultaneous addition of RTX, RF and serum (Figure 6A). RF blocked RTX-CDC independent of the time of addition (stepwise vs. simultaneous) (n = 3, P = 0.34). These data suggest that RF inhibits complement binding to the Fc portion of RTX after it has bound CD20. This interpretation was supported by the observation that RF had no effect on RTX binding to CD20, as measured by the lack of modulation of the ability of RTX to block the binding of fluorochrome-labeled anti-human CD20 (APC-Cy7) to Daudi cells (Figure 6B). Thus, RF does not limit the ability of RTX to bind CD20 on the B cell. These data suggest that RF inhibits C3b deposition and cytotoxicity by preventing C1q binding to the Fc portion of RTX.

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