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THU0029 Epratuzumab Influences BCR Signalling on TLR9 Pre-Activated B Cells by Targeting CD22
  1. N. Sieger1,2,
  2. S. Fleischer1,2,
  3. K. Reiter1,2,
  4. H. E. Mei1,2,
  5. A. Shock3,
  6. G. R. Burmester1,2,
  7. C. Daridon1,2,
  8. T. Dörner1,2
  1. 1Medicine/Rheumatology and Clinical Immunology, Charité University Medicine Berlin
  2. 2German Rheumatism Research Centre (DRFZ), Berlin, Germany
  3. 3UCB Pharma, Slough, United Kingdom


Background CD22, a transmembrane protein exclusively expressed on B cells, mediates migration by cell–cell interaction and negatively regulates B-cell receptor (BCR) signaling. CD22 acts as an inhibitory co-receptor of the BCR via de-phosphorylation of signaling molecules such as spleen tyrosine kinase (Syk) and subsequent phospholipase C (PLC)-γ2-triggered Ca2+ fluxes. The humanized anti-CD22 monoclonal antibody epratuzumab modulates adhesion molecule expression and B-cell migration in vitro. However, the potential of CD22 to regulate BCR signaling has not been fully delineated.

Objectives As toll-like receptor (TLR)-dependent activation of B cells represents an important pathway of B-cell hyperactivity in autoimmunity, our studies addressed whether epratuzumab might be able to modulate BCR signaling pathways in TLR9 pre-activated B cells or whether TLR9 activation abrogates the inhibitory effect of epratuzumab on the BCR signaling.

Methods Our study focused on the influence of epratuzumab on the B cells’ response to BCR alone or in combination with TLR9. The recruitment of CD22 to the BCR (CD79α) after epratuzumab incubation on B cells from healthy volunteers was analyzed by confocal microscopy. The in vitro effects of epratuzumab on BCR-induced signaling were evaluated by analyzing the phosphorylation status of BCR-signaling molecules, Syk and PLC-γ2 by flow cytometry with or without TLR9 pre-activation. The concentration of intracellular Ca2+ was monitored by flow cytometry after BCR stimulation. Finally, to evaluate the origin of the Ca2+ flux, extracellular Ca2+ was chelated with 1 mM ethylene glycol tetraacetic acid (EGTA).

Results B cells incubated with epratuzumab showed a specific co-localization of CD22 to the BCR-associated molecule CD79α on B cells. In addition, pre-treatment with epratuzumab led to a reduction in the MFI of phosphorylated Syk and PLC-γ2 induced by BCR stimulation compared with IgG1 isotype control. Similar results were observed when the cells were pre-incubated with F(ab’)2 fragment of epratuzumab, which excludes an inhibitory effect dependent on FcR signaling. Interestingly, the reduction of BCR induced kinase phosphorylation was demonstrated in both CD27- and CD27+ memory B cells. In addition, pre-activation of B cells by TLR9 did not circumvent the influence of epratuzumab on BCR signaling, as shown by a reduction in the MFI of phosphorylated Syk and PLC-γ2 in comparison to non-activated cells. Finally, a F(ab’)2 fragment of epratuzumab reduced BCR-induced calcium mobilization.

Conclusions Intracellular BCR signals can be modulated by CD22 ligation using epratuzumab, and pre-activation with TLR9 did not circumvent this effect. These data expand the potential mechanisms of action of epratuzumab.

Disclosure of Interest N. Sieger Grant/research support from: UCB Pharma, S. Fleischer Grant/research support from: UCB Pharma, K. Reiter Grant/research support from: UCB Pharma, H. Mei Grant/research support from: UCB Pharma, A. Shock Employee of: UCB Pharma, G. Burmester Grant/research support from: UCB Pharma, C. Daridon Grant/research support from: UCB Pharma, T. Dörner Grant/research support from: UCB Pharma, Consultant for: UCB Pharma

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