HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: jimmunol.0900632v1 183/1/749    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Pawluczkowycz, A. W. Articles by Taylor, R. P. PubMed PubMed Citation Articles by Pawluczkowycz, A. W. Articles by Taylor, R. P.

Binding of Submaximal C1q Promotes Complement-Dependent Cytotoxicity (CDC) of B Cells Opsonized with Anti-CD20 mAbs Ofatumumab (OFA) or Rituximab (RTX): Considerably Higher Levels of CDC Are Induced by OFA than by RTX¹

Andrew W. Pawluczkowycz^*, Frank J. Beurskens, Paul V. Beum^*, Margaret A. Lindorfer^*, Jan G. J. van de Winkel^,, Paul W. H. I. Parren and Ronald P. Taylor^2,*

^* Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908; Genmab, Utrecht, The Netherlands; and Immunotherapy Laboratory, Department of Immunology, University Medical Center, Utrecht, The Netherlands

The CD20 mAb ofatumumab (OFA) is more effective than rituximab (RTX) in promoting complement-dependent cytotoxicity (CDC) of B cells via the classical pathway (CP) of complement. CP activation is initiated by C1q binding to cell-bound IgG. Therefore, we examined the role of C1q in the dynamics of complement activation and CDC of B cell lines and primary cells from patients with chronic lymphocytic leukemia, reacted with OFA or RTX. C1q binding, complement activation, and colocalization of C1q with cell-bound mAbs were determined by flow cytometry and high-resolution digital imaging. C1q binds avidly to OFA-opsonized Raji and Daudi cells (K_D = 12–16 nM) and colocalizes substantially with cell-bound OFA. Cells opsonized with OFA undergo high levels of complement activation and CDC in C1q-depleted serum supplemented with low concentrations of C1q. Under comparable conditions, RTX-opsonized cells bind less C1q; in addition, even when higher concentrations of C1q are used to achieve comparable C1q binding to RTX-opsonized cells, less complement activation and CDC are observed. Greater CDC induced by OFA may occur because C1q is bound in close proximity and with high avidity to OFA, resulting in effective CP activation. Moreover, OFA binds to the small, extracellular CD20 loop, placing the mAb considerably closer to the cell membrane than does RTX. This may facilitate effective capture and concentration of activated complement components closer to the cell membrane, potentially shielding them from inactivation by fluid phase agents and promoting efficient generation of the membrane attack complex.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a research grant from Genmab.

² Address correspondence and reprint requests to Dr. Ronald P. Taylor, Department of Biochemistry and Molecular Genetics, P.O. Box 800733, University of Virginia, Charlottesville, VA 22908. E-mail address: rpt{at}virginia.edu

³ Abbreviations used in this paper: RTX, rituximab; Al, Alexa; CCP, complement control proteins; CDC, complement-dependent cytotoxicity; CLL, chronic lymphocytic leukemia; CP, classical pathway of complement; MESF, molecules of equivalent soluble fluorophore; NHS, normal human serum; OFA, ofatumumab, PI, propidium iodide.