Radioimmunotherapy (RIT) has emerged as an effective treatment for lymphoma, however the underlying mechanisms are poorly understood. We therefore investigated the relative contributions of antibody and targeted radiation to the clearance of tumor in vivo, using 2 different syngeneic murine B-cell lymphoma models. Although RIT with (131)I-anti-major histocompatibility complex class II (MHCII) was effective in targeting radiation to tumor, no improvement in survival was seen by escalating the radiation dose alone and there were no long-term survivors. In contrast, using the combination of (131)I anti-MHCII in the presence of unlabeled anti-idiotype (anti-Id), 100% prolonged disease-free survival was seen in both B-cell lymphoma models at the higher radiation dose. Using in vivo tracking we show that treatment with radiation plus anti-Id monoclonal antibody (mAb) results in a substantially greater reduction of splenic tumor cells than with either treatment alone. Prolonged survival could also be achieved using (131)I anti-MHCII plus the signaling anti-CD19 mAb. Furthermore, the ability of these anti-B-cell mAbs to improve survival with targeted radiotherapy appeared to correlate with their ability to initiate intracellular signal transduction. Together these data illustrate that using 1 mAb to target radiation to tumor and a second to induce cell signaling is an effective new strategy in RIT.

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

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