IgG1 antibodies with mutant Fc domains from selected clones were constructed with the antigen-binding fragment (Fab) of Rituxan (mAb to human CD20) and were expressed and purified from mammalian cells either in glycosylated form or as aglycosylated antibodies through the introduction of a T299L substitution that disrupts the Asn297X298-Thr299 (where X is any amino acid) glycosylation motif in the Fc (Supplementary Fig. and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings. Therapeutic monoclonal antibodies (mAbs) ameliorate disease by two mechanisms that involve the binding and resultant modulation of the function of proteins associated with pathophysiology and the recruitment of effector mechanisms dependent on the crystallizable fragment (Fc) regions of antibody domains; these functions mediate, either directly or indirectly, the neutralization and clearance of targeted substrates, as well as the programming of adaptive immunity1,2. Effector functions arise from the binding of the Fc domain of immunoglobulin G (IgG) to Fc receptors (FcRs) expressed on various leukocyte subsets and also from recruitment of the complement component C1q and the ensuing activation of the classical complement pathway. Human effector FcRs include, in addition to the well-characterized classical (type I) receptors (in humans, FcRI, FcRII, FcRIII and their isoforms), the lectin-like type II receptors (CD23 and CD209), TRIM21 and members of the FCRL family of receptors3,4. The recruitment and signaling of type I receptors via immunocomplexes (ICs) are responsible for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellmediated phagocytosis (ADCP), reactions that have been established clinically to contribute to the mechanism of action of many therapeutic antibodies5. Alternatively, activation of the classical complement pathway leads to target-cell clearance by two distinct processes6: first, direct cell lysis that results from insertion of the membrane attack complex into the cell membrane (complement-dependent cytotoxicity (CDC)); and second, the deposition of opsonins, such as C3b, that are covalently bound onto the cell surface and in turn are recognized by complement receptors (CRs) on effector cells. The Broussonetine A CRs activated by the deposited opsonins trigger complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP)6,7. Additionally, activation of the classical pathway has been Rabbit Polyclonal to NCOA7 established to stimulate B cell and T cell adaptive immune responses8. Determining in a quantitative way the relative roles of complementdependent and FcR-dependent effector mechanisms in mAb function is Broussonetine A critical for the development of improved therapeutics9,10. However, this has proven to be a very difficult problem to address experimentally, as evinced by the longstanding debate about the relative importance of complement in the clearance of CD20+ B cells by mAbs (such as rituximab (Rituxan)) to the B cellCspecific surface antigen CD20 (refs. 11,12). IgG isotypes capable of activating complement also bind to FcRs to varying degrees, especially after the formation of highly aggregated ICs on target cells or viruses13,14. As a result, it is not possible to distinguish, in the presence of serum, whether target-cell lysis by antibodies is dominated by Broussonetine A ADCC or CDCC and, similarly, whether phagocytosis is due to ADCP or CDCP. While ADCC and ADCP can be readily studied by well-established assays15, there is no straightforward manner with which to quantify the effect of CDCC and CDCP on target-cell clearance by mAbs. Because the C1qand FcR-binding sites on the Fc domain are proximal and partially overlap, amino-acid substitutions engineered to diminish the binding of FcRs also eliminate the recruitment of C1q and vice versa16,17. Among the cell-elimination pathways triggered by the classical complement pathway, CDC activity is by Broussonetine A far the easiest to measure and has been studied in great detail11,15. In contrast, apart from the results of some very early, qualitative studies from more than 40 years ago, with polyclonal antibodies18, practically nothing is known about the kinetics and magnitude of target-cell elimination by CDCC and CDCP or their importance in mAb function. In the.