Abstract
Abstract 1357
The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases, treating currently over 1000 cases of non-Hodgkin lymphoma (NHL), leukemias, Waldenström's macroglobulinemia, Sjögren's syndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action (MOA) remains poorly understood to date. Because epratuzumab reduces on average 35% of circulating B cells in patients, and has minimal antibody–dependent cell-mediated cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, we reason its therapeutic activity may not result completely from B-cell depletion; instead, ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Here we report for the first time that epratuzumab induces a substantial reduction of CD22 along with CD19, CD21, and CD79b, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or lupus patients, and of cells from three NHL lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Intriguingly, only CD22, but not others, was appreciably down-regulated by epratuzumab in isolated B cells, implicating additional cell-based factors are required.
PBMCs, from either healthy donors or lupus patients with flares, were incubated with epratuzumab (10 μg/mL) for 2 h or 18 h, and the relative surface expression levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Treatment of PBMCs from healthy donors with epratuzumab, but not an isotype control mAb, induced a marked down-regulation of CD22 (>80%), CD19 (>50%), CD21 (>50%) and CD79b (>30%) on the surface of B cells, with the stronger effect observed for CD27-negative B cells. The effect of epratuzumab could be observed after 2 h and increased moderately with overnight incubation (18 h). Moreover, substantial reduction of CD22 (>50%), but not others, was achieved with the F(ab')2 of epratuzumab generated from pepsin digestion. Similar results were observed for B cells in PBMCs of lupus patients. In the absence of PBMCs, treatment of NHL cell lines (Daudi, Raji and Ramos) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (<15%) suppression of CD19 and CD21. When these NHL cells (1×105 cells) were mixed with PBMCs (1×106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Importantly, in all these experiments, there was no evidence of depletion in B cells, whether malignant or not; nor had epratuzumab induced an increase in apoptotic (Annexin V+) B cells in PBMCs from any of 4 donors, compared to the untreated (22.9 ± 1.8% versus 22.1 ±1.2%; P=0.6864).
This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The prominent down-regulation of CD19, CD21, and CD79b by epratuzumab is not only Fc-dependent, but also requires further engagement with certain effector cells present in PBMCs. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in autoimmune diseases, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold.
Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.
Author notes
Asterisk with author names denotes non-ASH members.
