Complement Activation on B Lymphocytes Opsonized with Rituximab or Ofatumumab Produces Substantial Changes in Membrane Structure Preceding Cell Lysis

Abstract

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties

“…In addition to the difference in binding sites between ofatumumab and rituximab, studies have suggested that ofatumumab dissociates more slowly from the cell surface than rituximab 35 and exhibits greater CDC activity than rituximab in various B cell lines. 35,48,75,77 Furthermore, CDC by ofatumumab was found to be less dependent on the cell-surface density of CD20 than CDC by rituximab. 48 The differential action of ofatumumab on the complement has been supported by direct visualization of complement-mediated cell killing obtained using spinningdisk confocal microscopy.…”

“…This could be expected to facilitate the deposition of activated complement on the cell surface and hence the amplification of the complement response. 77 However, the impact of this is unclear as the CD20 extracellular loop is very small compared with the size of an antibody so that the antibody-binding domain of CD20 is already membrane-proximal. In addition to the difference in binding sites between ofatumumab and rituximab, studies have suggested that ofatumumab dissociates more slowly from the cell surface than rituximab 35 and exhibits greater CDC activity than rituximab in various B cell lines.…”

“…48 The differential action of ofatumumab on the complement has been supported by direct visualization of complement-mediated cell killing obtained using spinningdisk confocal microscopy. 77 Compared with rituximab, ofatumumab has been shown to be more active in both the deposition of complement and in causing morphologic effects induced by the membrane attack complexes of complement, namely blebbing (the formation of bulges in the cell membrane) and the creation of long, thin 'streamer' structures that extend from the cell membrane. Other data, however, have suggested that the preclinical activity of ofatumumab and rituximab are similar, demonstrating comparable levels of CDC, ADCC, whole blood B cell depletion and antitumor activity in preclinical assays and models.…”

Ofatumumab

“…Further in-vitro experiments indicated ofatumumab is effective in killing B-cells extracted from CLL patients. 14 …”

“…Hybridoma that secreted human IgG1 anti-CD20 antibodies were extracted from successfully immunized mice. 13,14 Using genetic engineering techniques, heavy and light chain genes from one human anti-CD20 cell line, 2F2, were transfected into a murine myeloma cell line (NS/O) for production of ofatumumab.…”