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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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RTX-CDC varies as a function of patient sera and correlates with RF levels. A. RTX-CDC using Daudi cell line with 1% human sera stratified by disease state: healthy donors, non-RA disease, seronegative RA (RF-), and seropositive RA (RF+). Data represent the mean cell death obtained with 15 sera in each category except for RF+ (n = 40). B, C, D. RTX-CDC cell death declines with increasing titer of RF, with a stronger correlation with IgM RF. Error bars represent mean standard error.
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Figure 2: RTX-CDC varies as a function of patient sera and correlates with RF levels. A. RTX-CDC using Daudi cell line with 1% human sera stratified by disease state: healthy donors, non-RA disease, seronegative RA (RF-), and seropositive RA (RF+). Data represent the mean cell death obtained with 15 sera in each category except for RF+ (n = 40). B, C, D. RTX-CDC cell death declines with increasing titer of RF, with a stronger correlation with IgM RF. Error bars represent mean standard error.

Mentions: We hypothesized that the presence of RF in human sera would enhance RTX-CDC. To investigate this, we measured Daudi cell death using sera from four different populations: healthy donors (n = 15), non-RA patients (n = 15), seronegative RA patients (n = 15), and seropositive RA patients (n = 40). Using limiting serum concentrations (1%), the average cell death in sera from healthy donors and seropositive RA donors was very similar (54% and 47%, respectively, P = 0.28) (Figure 2A), though greater variability was seen using sera from seropositive patients. Interestingly, the RTX-CDC seen with non-RA patients and seronegative RA patients was significantly higher (P < 0.0001). While it was not surprising that sera from patients with systemic inflammation had higher RTX-CDC than healthy donors, the reduction in RTX-CDC and the wide variability of RTX-CDC seen with sera from seropositive RA patients was completely unexpected. This apparent incongruity became clear upon finding that stratification of RTX-CDC as a function of RF titer clearly showed an inverse correlation (Figure 2B-D). The correlation held true with IgA RF (R2 = 0.18; P < 0.006), but was stronger for IgM RF (R2 = 0.45; P < 0.0001) (Figure 3).


A rheumatoid factor paradox: inhibition of rituximab effector function.

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

RTX-CDC varies as a function of patient sera and correlates with RF levels. A. RTX-CDC using Daudi cell line with 1% human sera stratified by disease state: healthy donors, non-RA disease, seronegative RA (RF-), and seropositive RA (RF+). Data represent the mean cell death obtained with 15 sera in each category except for RF+ (n = 40). B, C, D. RTX-CDC cell death declines with increasing titer of RF, with a stronger correlation with IgM RF. 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 2: RTX-CDC varies as a function of patient sera and correlates with RF levels. A. RTX-CDC using Daudi cell line with 1% human sera stratified by disease state: healthy donors, non-RA disease, seronegative RA (RF-), and seropositive RA (RF+). Data represent the mean cell death obtained with 15 sera in each category except for RF+ (n = 40). B, C, D. RTX-CDC cell death declines with increasing titer of RF, with a stronger correlation with IgM RF. Error bars represent mean standard error.
Mentions: We hypothesized that the presence of RF in human sera would enhance RTX-CDC. To investigate this, we measured Daudi cell death using sera from four different populations: healthy donors (n = 15), non-RA patients (n = 15), seronegative RA patients (n = 15), and seropositive RA patients (n = 40). Using limiting serum concentrations (1%), the average cell death in sera from healthy donors and seropositive RA donors was very similar (54% and 47%, respectively, P = 0.28) (Figure 2A), though greater variability was seen using sera from seropositive patients. Interestingly, the RTX-CDC seen with non-RA patients and seronegative RA patients was significantly higher (P < 0.0001). While it was not surprising that sera from patients with systemic inflammation had higher RTX-CDC than healthy donors, the reduction in RTX-CDC and the wide variability of RTX-CDC seen with sera from seropositive RA patients was completely unexpected. This apparent incongruity became clear upon finding that stratification of RTX-CDC as a function of RF titer clearly showed an inverse correlation (Figure 2B-D). The correlation held true with IgA RF (R2 = 0.18; P < 0.006), but was stronger for IgM RF (R2 = 0.45; P < 0.0001) (Figure 3).

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