Background/Purpose:

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.

Methods:

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.

Results:

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.

Conclusion:

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.

References:

1. Sieger N. Arth Rheum 2013;65:770

2. Rossi E. Blood 2013;122:3020

SL and SF contributed equally to this work.

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ACR Meeting Abstracts - 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/