Background/Purpose:

Epratuzumab is a monoclonal antibody targeting CD22 that is currently in phase III clinical trials in systemic lupus erythematosus (SLE) patients. CD22 is found almost exclusively on B cells and in vitro culture with epratuzumab down-regulates B cell receptor (BCR)-dependent cell signaling and B cell activation events. The objective of this study was to investigate epratuzumab-dependent changes in phosphoprotein signals in activated B cells using a recently developed LC-MS/MS methodology (TIQUAS^TM or Targeted Quantification of Cell Signalling).

Methods:

B cells were purified from human tonsils (n=8 donors) by mechanical homogenisation followed by Ficoll-Hypaque gradient centrifugations. T cells were depleted by E-rosetting using 2 aminoethylisothiouronium bromide (AET) treated sheep red blood cells. The resulting B cells were counted and analysed by flow cytometry for purity, typically B cells preparations were >90%pure. 4×10⁷ cells B cells were then stimulated through the BCR using anti-IgM for 2 minutes with or without prior pre-incubation with epratuzumab or IgG1 isotype control for 1 hour. Immediately following stimulation the cells were harvested in ice cold PBS buffer containing phosphatase inhibitors, pelleted by centrifugation and lysed in a urea based lysis buffer containing phosphatase inhibitors. The cell lysates were sonicated on ice and quantitated by BCA protein assay. 500μg of cell extracts were subject to protease digestion and TiO2 phosphopeptide enrichment. LC-MS-MS phosphoproteomic analysis was performed using a label-free quantification strategy. A mixed linear effect statistical model was applied to the quantitation output which comprised data for 3825 distinct phosphorylation sites. The phosphopeptide data set was imported into Ingenuity Pathway Analysis (IPA) and mapped to canonical pathways for further analysis.

Results:

This analysis was able to identify statistically significantly regulated phosphorylation sites in response to pre-incubation with epratuzumab using adjusted p- and q- threshold values of <0.05, although the most significant changes generally showed no more than a 2-fold difference from signals induced by BCR stimulation alone. Among the changes observed were BCR-specific downstream signals on a broad range of protein family types: adaptor proteins (SHC1), kinases (ERK1/2, p38delta), phosphatases (SHIP1), histones (H1) and transcription factors (NFAT). Additionally, there was down-modulation of the phosphorylation of Ser717 on CD22 itself.

Conclusion:

Pre-incubation of human tonsil-derived B cells with epratuzumab induced discrete but statistically significant changes in phosphoprotein signals after BCR activation. Such observations may enable a better understanding of how epratuzumab modulates B cell functions in vitro and possibly in patients with SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/phosphoprotein-changes-induced-with-epratuzumab-an-antibody-targeting-cd22-on-b-cells/