Abstract
We examined the kinetics and mechanisms by which monoclonal antibodies (mAbs) utilize complement to rapidly kill targeted cancer cells. Based on results from flow cytometry, confocal microscopy and high-resolution digital imaging experiments, the general patterns which have emerged reveal cytotoxic activities mediated by substantial and lethal Ca2+ fluxes. The Ca2+ fluxes are common to the reported pathways that have been utilized by other toxins in killing nucleated cells. These reactions terminate in very high levels of cell killing, and based on these considerations, we suggest additional strategies to further enhance mAb-based targeting of cancer with complement.
Highlights
Complement was first described and characterized by Bordet more than 100 years ago [1]; he demonstrated it to be a heat-labile factor in serum that promoted destruction of bacteria and/or hemolysis of erythrocytes, each opsonized with the antibodies in immune sera
In order to concentrate on mechanisms, we have examined multiple individual steps in the complement-dependent cytotoxicity (CDC) reaction that start with monoclonal antibodies (mAbs) binding and end with cell death in a continuously monitored reaction mediated by Food and Drug
Ca2+ concentration in the cytoplasm and mitochondria of the cell [16]; tetramethylrhodamine methyl ester (TMRME), which is highly fluorescent only in viable mitochondria [16]; mAb aE11, specific for membrane attack complex (MAC)-associated C9 deposited on cells [38]; and vital fluorescent dyes such as propidium iodide (PI), 7-aminoactinomycin D (7AAD) and TO-PRO-3 which enter dead permeabilized cells and, upon staining nuclear DNA, become highly fluorescent, providing reliable markers for cell death and successful CDC [16,19,22]
Summary
Complement was first described and characterized by Bordet more than 100 years ago [1]; he demonstrated it to be a heat-labile factor in serum that promoted destruction (lysis) of bacteria and/or hemolysis of erythrocytes, each opsonized with the antibodies in immune sera. The traditional view of the mechanism by which C mediates the killing of antibody-opsonized cells was based on classic experiments that focused on C-mediated lysis of non-nucleated sheep erythrocytes that were first opsonized with polyclonal rabbit antibodies before they were brought into contact with a source of C and incubated for a considerable period of time at 37 ◦ C to promote hemolysis [6,7,8]. The results of these studies led to the concept that insertion of the membrane attack complex (MAC). This model system has proven to be invaluable for dissecting out and identifying virtually all of the key components of C, including pathways, activating factors and inhibitors
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