Background CD22 is a trans-membrane protein exclusively expressed on B cells which regulates adhesion/migration and inhibits cellular responses to B-cell receptor (BCR) stimulation. Spleen tyrosine kinase (Syk) and subsequent phospholipase C (PLC)-γ2-triggered Ca2+ fluxes are rapidly activated after BCR stimulation. CD22 binds specifically to α-2,6-sialic acid residues present on a broad number of proteins, including CD22 itself. Sialo-interactions appear to be crucial for optimal function of CD22 and can occur in cis (to ligands on the same cell surface) or trans (to ligands on neighboring cells).
Objectives The humanized anti-CD22 monoclonal antibody epratuzumab, currently being tested in clinical trials, modulates adhesion molecule expression and B cell migration in vitro however, the potential of CD22-ligation with cis and trans sialic acid to regulate BCR signaling has not been delineated.
Methods In vitro effects of epratuzumab on BCR-induced signaling were evaluated by analyzing the phosphorylation of BCR-signaling molecules (Syk/PLC-γ2) and Ca2+ flux. The influence of epratuzumab on the phosphorylation of the BCR signaling molecules Syk and PLC-γ2 was studied in vitro by flow cytometry in PBMCs from healthy donors. To investigate the effect of sialo-interaction on the effects by epratuzumab, PBMCs were treated with neuraminidase (to remove sialic acic) and BCR induced phosphorylation (Syk and PLC-γ2) was detected with or without epratuzumab incubation. To monitor the concentration of intracellular Ca2+, B cells pre-loaded with Indo-1AM were incubated with or without F(ab’)2-epratuzumab for 60 minutes at 37°C and were subsequently stimulated with F(ab’)2 anti-IgM/IgG. Intracellular Ca2+ concentrations were monitored by flow cytometry over 10 minutes after activation.
Results Epratuzumab inhibited Syk and PLC-γ2 phosphorylation after in vitro BCR stimulation by 23±9% and 33±5, respectively. Interestingly, preventing sialo-interactions of CD22 partially reduced the inhibitory effect of epratuzumab on Syk and PLC-γ2 phosphorylation to 12±6% and 22±4%, respectively. The inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ memory B cells and appeared to be independent of FcR signaling since a F(ab’)2 fragment of epratuzumab induced comparable results to the full antibody. In addition, a F(ab’)2 fragment of epratuzumab reduced the BCR-induced calcium flux. These data are consistent with the potential of targeting CD22 to raise the threshold of BCR activation which also provides additional control of B cell function.
Conclusions Intracellular BCR signals can be partially inhibited by CD22 ligation with epratuzumab, providing further understanding of targeting CD22 biology in human autoimmune diseases.
This study was supported by Sonderforschungsbereich 650 and DFG491/7-1 and in part by UCB Pharma Inc.
Nitschke L. The role of CD22 and other inhibitory co-receptors in B-cell activation. Current opinion in immunology 2005;17:290–7.
Daridon C, Blassfeld D, Reiter K, et al. Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus. Arthritis Research & Therapy 2010;12:R204.
Disclosure of Interest N. Sieger: None Declared, S. Fleischer: None Declared, H. Mei: None Declared, K. Reiter: None Declared, A. Shock Employee of: UCB Pharma inc., G. Burmester: None Declared, C. Daridon: None Declared, T. Dörner Grant/Research support from: Immunomedics
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