title: Extensive crosslinking of CD22 by epratuzumab triggers BCR signaling and caspase-dependent apoptosis in human lymphoma cells.
abstract: Epratuzumab has demonstrated therapeutic activity in patients with non-Hodgkin lymphoma, acute lymphoblastic leukemia, systemic lupus erythematosus, and Sjögren's syndrome, but its mechanism of affecting normal and malignant B cells remains incompletely understood. We reported previously that epratuzumab displayed in vitro cytotoxicity to CD22-expressing Burkitt lymphoma cell lines (Daudi and Ramos) only when immobilized on plates or combined with a crosslinking antibody plus a suboptimal amount of anti-IgM (1 μg/mL). Herein, we show that, in the absence of additional anti-IgM ligation, extensive crosslinking of CD22 by plate-immobilized epratuzumab induced intracellular changes in Daudi cells similar to ligating B-cell antigen receptor with a sufficiently high amount of anti-IgM (10 μg/mL). Specifically, either treatment led to phosphorylation of CD22, CD79a and CD79b, along with their translocation to lipid rafts, both of which were essential for effecting caspase-dependent apoptosis. Moreover, such immobilization induced stabilization of F-actin, phosphorylation of Lyn, ERKs and JNKs, generation of reactive oxygen species (ROS), decrease in mitochondria membrane potential (Δψm), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-xl and Mcl-1. The physiological relevance of immobilized epratuzumab was implicated by noting that several of its in vitro effects, including apoptosis, drop in Δψm, and generation of ROS, could be observed with soluble epratuzumab in Daudi cells co-cultivated with human umbilical vein endothelial cells. These results suggest that the in vivo mechanism of non-ligand-blocking epratuzumab may, in part, involve the unmasking of CD22 to facilitate the trans-interaction of B cells with vascular endothelium.
authors.affiliation: a Immunomedics, Inc. ; Morris Plains , NJ USA.
authors.forename: Chien-Hsing
authors.initials: CH
authors.lastname: Chang
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Pankaj
authors.initials: P
authors.lastname: Gupta
authors.forename: David M
authors.initials: DM
authors.lastname: Goldenberg
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D002051:Burkitt Lymphoma / Q000188:drug therapy / Q000276:immunology*; D020169:Caspases / Q000276:immunology*; D045744:Cell Line, Tumor; D006801:Humans; D007152:Immunologic Capping / Q000187:drug effects*; D021962:Membrane Microdomains / Q000276:immunology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D021381:Protein Transport / Q000187:drug effects / Q000276:immunology; D011947:Receptors, Antigen, B-Cell / Q000037:antagonists & inhibitors* / Q000276:immunology; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000037:antagonists & inhibitors* / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; C497867:CD22 protein, human; D009363:Neoplasm Proteins; D011947:Receptors, Antigen, B-Cell; D051918:Sialic Acid Binding Ig-like Lectin 2; C448700:epratuzumab; D020169:Caspases
keywords: 488-annexin V, Alexa Fluor 488-conjugated annexin V; 7-AAD, 7-aminoactinomycin D, Syk, spleen tyrosine kinase; Anti-IgM, F(ab’)2 fragment of affinity-purified goat anti-human IgM, Fc5μ fragment; BCR; BCR, B-cell antigen receptor; BSA, bovine serum albumin; CD22; CM-H2DCF-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DNP, 2,4-dinitrophenyl; EC, endothelial cells; ERKs, extracellular signal-regulated kinases; FBS, fetal bovine serum; FITC-DNase I, fluorescein isothiocyanate-conjugated DNase I; GAH, F(ab′)2 fragment of affinity-purified goat anti-human IgG Fcγ fragment-specific; HUV-EC; HUV-EC, human umbilical vein endothelial cells; ITIM, immunoreceptor tyrosine-based inhibition motif; JNKs, c-Jun N-terminal kinases; JP, jasplakinolide; LatB, latrunculin B; Lyn, Lck/Yes novel tyrosine kinase; MAP kinases, mitogen-activated protein kinases; MTS, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PLCγ2, phospholipase C, isotype gamma 2; ROS, reactive oxygen species; Rhodamine-anti-IgG, rhodamine-conjugated F(ab′)2 fragment of affinity-purified goat anti-human IgG, F(ab′)2 fragment-specific; TMRE/tetramethylrhodamine/ethyl ester; epratuzumab; human B-cell lymphoma; immobilized; mIgM, membrane IgM; Δψm, mitochondria membrane potential