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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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Inhibition of RTX-CDC is mediated by RF. A. Mixing studies using seronegative (RF-) and seropositive (RF+) sera demonstrates inhibition of CDC by RF+ sera, indicating that an inhibitor, not a factor deficiency, decreases CDC. B. Addition of monoclonal IgM RF markedly inhibits RTX-CDC by NHS. C. Addition of monoclonal IgA RF markedly inhibits RTX-CDC by NHS. Graphs represent average of three experiments; error bars represent mean standard error.
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Figure 4: Inhibition of RTX-CDC is mediated by RF. A. Mixing studies using seronegative (RF-) and seropositive (RF+) sera demonstrates inhibition of CDC by RF+ sera, indicating that an inhibitor, not a factor deficiency, decreases CDC. B. Addition of monoclonal IgM RF markedly inhibits RTX-CDC by NHS. C. Addition of monoclonal IgA RF markedly inhibits RTX-CDC by NHS. Graphs represent average of three experiments; error bars represent mean standard error.

Mentions: One possible mechanism for this observation was the possible association of RF titer with reductions in serum complement. If true, a mixing study with normal human serum would be expected to correct the reduction in RTX-CDC. The mixing of 1% serum from a seropositive patient with those from seronegative patients showed the opposite result: RF+ sera inhibited RTX-CDC (n = 3, Figure 4A). Thus, the decreased RTX-CDC associated with RF+ sera operated in trans to block RTX-CDC.


A rheumatoid factor paradox: inhibition of rituximab effector function.

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

Inhibition of RTX-CDC is mediated by RF. A. Mixing studies using seronegative (RF-) and seropositive (RF+) sera demonstrates inhibition of CDC by RF+ sera, indicating that an inhibitor, not a factor deficiency, decreases CDC. B. Addition of monoclonal IgM RF markedly inhibits RTX-CDC by NHS. C. Addition of monoclonal IgA RF markedly inhibits RTX-CDC by NHS. Graphs represent average of three experiments; 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 4: Inhibition of RTX-CDC is mediated by RF. A. Mixing studies using seronegative (RF-) and seropositive (RF+) sera demonstrates inhibition of CDC by RF+ sera, indicating that an inhibitor, not a factor deficiency, decreases CDC. B. Addition of monoclonal IgM RF markedly inhibits RTX-CDC by NHS. C. Addition of monoclonal IgA RF markedly inhibits RTX-CDC by NHS. Graphs represent average of three experiments; error bars represent mean standard error.
Mentions: One possible mechanism for this observation was the possible association of RF titer with reductions in serum complement. If true, a mixing study with normal human serum would be expected to correct the reduction in RTX-CDC. The mixing of 1% serum from a seropositive patient with those from seronegative patients showed the opposite result: RF+ sera inhibited RTX-CDC (n = 3, Figure 4A). Thus, the decreased RTX-CDC associated with RF+ sera operated in trans to block RTX-CDC.

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