Apparent modulation of CD20 by rituximab: an alternative explanation

Abstract

We read with interest the paper from Jilani et al 1 in which rituximab treatment appeared to down-modulate CD20 expression through a combination of internalization and RNA regulation. The result is unexpected because previous studies had shown that CD20 is not modulated by monoclonal antibody (mAb) treatment, 2-4 even in vivo. 5 The study by Jilani et al used an anti-mouse immunoglobulin polyclonal antibody (Ab) that binds to the mouse V regions in rituximab. This reagent will bind to rituximab, while the chimeric antibody is coated onto CD20 ϩ cells. We are concerned that the apparent loss of binding reported may be due to factors other than loss of CD20 expression-specifically blocking by normal immunoglobulin.As part of our ongoing CD20 studies, we have developed a mouse anti-idiotype (Id) mAb (2A4) specific for the V regions of rituximab and its parent Ab 2B8 (M.S.C. and M.C.B., manuscript submitted, March 2004). This reagent binds rituximab or 2B8 while it is coated onto CD20 ϩ cells ( Figure 1A). Using this highly specific reagent we can find no evidence that CD20 is down-modulated on malignant B cells in either the presence or absence of plasma ( Figure 1B). Using the anti-Id mAb (MB2A4), together with a human Fc␥-specific mAb, we examined the ability of rituximab and its murine counterpart 2B8 to bind to CD20 on Raji cells and fresh B-cell chronic lymphocytic leukemia (B-CLL) cells with and without plasma. In these experiments, cells were incubated for 2 hours at 37°C as described by Jilani et al, before the cells were washed twice by centrifugation and stained for surface CD20 mAb. Interestingly, when using fluorescein isothiocyanate (FITC)-conjugated anti-Fc␥ mAb, our

Impact of low-dose rituximab on splenic B cells in ABO-incompatible renal transplant recipients

“…In the first, since CD79a expression is known to precede CD19 or CD20 expression in the earliest differentiation events of B-cell ontogeny [15,16], it is possible that the CD79a + CD20 ) immature B cells, which are not susceptible to RIT, were residual in the spleen. Previous studies [8,9] [17,18] have demonstrated the possible internalization of CD20 epitopes after RIT administration; it has also been suggested that RIT can mask CD20 epitopes, resulting in false negative expression of CD20 in FACS analysis of B cells in peripheral blood, as FACS analysis can detect only extracellular epitopes. However, B cells express not only extracellular CD20 epitopes but also intracellular CD20 epitopes.…”

Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection

“…Furthermore, we have reported that CD20 did not substantially modulate from Raji BL cells or a primary CLL sample in a short-term 2-hour culture. 45 To clarify these apparent discrepancies, we determined to study modulation on a variety of B-cell targets. Michel and Mattes have previously demonstrated the internalization of CD20 on certain human B-cell lines after mAb engagement but found it to be slow with little internalization until 18 hours.…”

International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia

“…10,15,16 There have been a wide variety of MRD flow assays reported and most rely to some extent on assessment of CD20 expression, which may be compromised during rituximab therapy. [19][20][21] In order to allow direct comparison of the different approaches, it is necessary to develop a consensus reagent set that can be used in peripheral blood and bone marrow at all stages of the disease and independent of the therapeutic approach.…”