Abstract
IntroductionEpratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin's lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. Treatment of SLE patients with epratuzumab leads to a reduction of circulating CD27negative B-cells, although epratuzumab is weakly cytotoxic to B-cells in vitro. Therefore, potential effects of epratuzumab on adhesion molecule expression and the migration of B-cells have been evaluated.MethodsEpratuzumab binding specificity and the surface expression of adhesion molecules (CD62L, β7 integrin and β1 integrin) after culture with epratuzumab was studied on B-cell subsets of SLE patients by flow cytometry. In addition, in vitro transwell migration assays were performed to analyze the effects of epratuzumab on migration towards different chemokines such as CXCL12, CXCL13 or to CXCR3 ligands, and to assess the functional consequences of altered adhesion molecule expression.ResultsEpratuzumab binding was considerably higher on B-cells relative to other cell types assessed. No binding of epratuzumab was observed on T-cells, while weak non-specific binding of epratuzumab on monocytes was noted. On B-cells, binding of epratuzumab was particularly enhanced on CD27negative B-cells compared to CD27positive B-cells, primarily related to a higher expression of CD22 on CD27negative B-cells. Moreover, epratuzumab binding led to a decrease in the cell surface expression of CD62L and β7 integrin, while the expression of β1 integrin was enhanced. The effects on the pattern of adhesion molecule expression observed with epratuzumab were principally confined to a fraction of the CD27negative B-cell subpopulation and were associated with enhanced spontaneous migration of B-cells. Furthermore, epratuzumab also enhanced the migration of CD27negative B-cells towards the chemokine CXCL12.ConclusionsThe current data suggest that epratuzumab has effects on the expression of the adhesion molecules CD62L, β7 integrin and β1 integrin as well as on migration towards CXCL12, primarily of CD27negative B-cells. Therefore, induced changes in migration appear to be part of the mechanism of action of epratuzumab and are consistent with the observation that CD27negative B-cells were found to be preferentially reduced in the peripheral blood under treatment.
Highlights
Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin’s lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive
CD22, a 140 kDa transmenbrane type 1 protein, called Sialic acid-binding Ig-like lectin 2 (Siglec-2) or B-lymphocyte cell adhesion molecule (BL-CAM), is a member of the Siglec family that binds to a2-6-linked sialic acids on glycoproteins
Migration towards CXCL12 (50 nM) or CXCL13 (250 nM) (B-cell homing chemokine, BLC or B-cell attracting chemokine 1, BCA1) or to a mix of CXCR3 ligands (CXCL9 (250 nM), CXCL10 (300 nM) and CXCL11 (10 nM)) were studied by adding the different chemokines to the lower chamber in RPMI 1640 supplemented with 0.5% (w/v) bovine serum albumin (BSA) as described previously [26]
Summary
Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin’s lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. In this context, epratuzumab, a humanized monoclonal IgG1 antibody (mAb) that targets the B-cell surface molecule CD22, has been explored in an early clinical trial [2] and more recently in a phase IIb randomized clinical study which showed a treatment advantage with epratuzumab over placebo of around 25% at week 12 [3]. CD22, a 140 kDa transmenbrane type 1 protein, called Sialic acid-binding Ig-like lectin 2 (Siglec-2) or B-lymphocyte cell adhesion molecule (BL-CAM), is a member of the Siglec family that binds to a2-6-linked sialic acids on glycoproteins These ligands for CD22 are widely expressed on different cell types [4] (co called trans glycoprotein ligands) including B-cells (where CD22 will bind cis glycoprotein ligands). Stathish et al described the expression of CD22 on murine primary T-cells [8], CD22 has not been detected on human T-cells and monocytes [4]
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