Date: Monday, November 9, 2015
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Epratuzumab, a humanized monoclonal antibody targeting CD22, is currently in phase 3 clinical trials in patients with systemic lupus erythematosus (SLE). Previous work suggests epratuzumab down-modulates B cell receptor (BCR) function.1,2 Inhibitory co-receptors of the BCR complex, such as CD22 and the inhibitory Fc receptor CD32B, prevent overstimulation of B cells by signaling through tyrosine (Tyr) based inhibitory motifs (ITIMs). Phosphorylation of Tyr residues on CD22 results in the recruitment of adaptor and inhibitory signaling molecules eg. Tyr822 binds SHP-1 and Tyr807 binds Grb2. In contrast CD32B contains a single ITIM motif (Tyr292) that down-regulates activating signals. In this study the effects of epratuzumab on the phosphorylation of inhibitory motifs within CD22 and CD32B were assessed.
Fluorescence-activated cell sorting (FACS) was conducted on peripheral blood mononuclear cells from healthy donors. After addition of epratuzumab (10 µg/ml; n=6) or anti-BCR (12 µg/ml; n=8) for various durations, cells were fixed, permeabilized and analyzed using a phospho-Tyr822 antibody and B cell markers CD19/CD20/CD27. Confocal microscopy assessed co-localization of CD22 with SHP-1 (n=3) using specific fluorescently-labelled antibodies. For immunoprecipitation (IP) experiments, B cells from human tonsils or Daudi B cells were treated ± epratuzumab or anti-BCR (both at 10 mg/mL), lysed and IP’d with epratuzumab or CD32B antibodies. Captured proteins were identified by Western blotting. Phosphorylated Tyr residues were detected using phospho-specific antibodies against p-Tyr807 and p-Tyr822 on CD22, and p-Tyr292 on CD32B. Signals were detected using HRP-linked secondary antibodies.
FACS experiments showed that anti-BCR induced a rapid (<5 min) increase in CD22 Tyr822 phosphorylation in both naïve (CD27-) and memory (CD27+) B cells. Epratuzumab stimulated Tyr822 phosphorylation in a similar time frame, albeit to a lesser extent than anti-BCR (52% less phosphorylation). In keeping with this, epratuzumab induced SHP-1 co-localization with CD22. An increase in CD22 Tyr822 phosphorylation with epratuzumab was detected in IP experiments in Daudi cells. These experiments showed increased CD22 Tyr807 phosphorylation at early (<10 min) time points in tonsil B cells and Daudi cells, although to a lesser extent than anti-BCR. Finally, epratuzumab induced a modest increase in CD32B ITIM Tyr292 phosphorylation on B cells peaking at approximately 10 min.
Epratuzumab directly induced phosphorylation of inhibitory ITIM motifs within key negative regulatory B cell molecules. On CD22, these included Tyr822, with a concomitant increase in SHP-1 co-localization and Tyr807, both of which would inhibit BCR-driven signaling. Finally, epratuzumab induced Tyr292 phosphorylation on the inhibitory Fc receptor CD32B, potentially dampening a hyper-reactive B cell response. Overall, the data provide further evidence that epratuzumab down-modulates B cell activation events, of potential relevance to the treatment of SLE patients with epratuzumab.
1. Sieger N. Arth Rheum 2013;65:770
2. Rossi E. Blood 2013;122:3020
SL and SF contributed equally to this work.
To cite this abstract in AMA style:Lumb S, Fleischer SJ, Daridon C, Maloney A, Shock A, Dorner T. Epratuzumab, a Monoclonal Antibody Targeting CD22 on B Cells, Stimulates the Phosphorylation of Upstream Inhibitory Signals of the B Cell Receptor [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/epratuzumab-a-monoclonal-antibody-targeting-cd22-on-b-cells-stimulates-the-phosphorylation-of-upstream-inhibitory-signals-of-the-b-cell-receptor/. Accessed August 9, 2020.
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