1261 - Development of a Whole Blood Assay to Determine Rituximab Mediated Complement Dependent Cytotoxicity of B Lymphocytes

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Jonathan D. Jones, Dartmouth-Hitchcock Medical Center, Lebanon, NH, B. JoNell Hamilton, Dartmouth College, Lebanon, NH, Whitney Hilton, Dartmouth Hitchcock Med Ctr, Lebanon, NH and William F. C. Rigby, Dartmouth-Hitchcock Med Ctr, Lebanon, NH
Presentation Number: 1261


Rituximab (RTX) is a monoclonal antibody targeting CD20, a marker found exclusively on B lymphocytes. RTX has shown efficacy in treating rheumatoid arthritis (RA). Although RTX depletes circulating B cells in all patients, less than half of RA patients achieve a high level of response. Moreover, the clinical response to RTX trails peripheral blood B cell depletion by weeks, if not months. Studies have suggested that synovial B cell depletion is important for clinical response, but the mechanism of B cell depletion in this compartment is not clear, particularly for non-circulating, resident synovial B cells. One possibility is that for clinical responses to occur, RTX must mediate synovial depletion of resident B cells via complement-dependent cytotoxicity (CDC) of B lymphocytes. By extension of this, non-responders fail to deplete synovial B cells by CDC. We report our development of a novel whole blood assay of RTX-CDC using hirudin anticoagulation and measurement of variable activity in normal volunteer donors.


Peripheral blood was drawn from healthy donors using hirudin, heparin or EDTA as an anticoagulant. Optimum hirudin concentration was found to be 50 μg/ml. Rituximab was added at varying concentrations and for various times and the percentage of CD19+ B cells as a function of all CD45+ cells determined by flow cytometry (FACScalibur, BD Biosciences).


Using hirudin as an anticoagulant, we observed a rapid (15 minute) reduction in CD19 B cells with RTX concentrations as low as 0.01 μg/ml. No depletion was seen with RTX treatment when heparin or EDTA was employed as an anticoagulant, suggesting complement dependence. The role of complement activation was confirmed as B cell depletion was blocked by the addition of compstatin, which prevents the cleavage of C3 (Figure 1). The average percentage of B cell depletion was 35%, with an inter-donor variability of 11% to 60%. Only a portion of B cells deplete in this assay, and there is marked inter-donor heterogeneity of B cell depletion, findings which we are continuing to explore. These findings likely underlie the variability seen in RA response to rituximab.


We have developed an assay that measures RTX mediated CDC of B cells. This assay presents a novel approach to assess the heterogeneity of RTX response, and will allow for increased understanding of the mechanisms of RTX effect. This may account for the variable success of RTX in various rheumatologic diseases, and has the potential to lead to a model to predict a priori RTX response. Additionally, these studies may provide insight into the success of other monoclonal antibodies currently in development.

Figure 1 Addition of compstatin, a C3 inhibitor, blocked the reduction in CD19+ B cells following RTX addition, confirming the role of complement activation in the observed B lymphocyte depletion.


Keywords: B cells and rituximab

Disclosure: J. D. Jones, None; B. J. Hamilton, None; W. Hilton, None; W. F. C. Rigby, Genentech, Inc., 2, Genentech, Inc., 5, Genentech, Inc., 8 .