How Do mAbs Make Use of Complement to Kill Cancer Cells? The Role of Ca2+

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.

Complement-Dependent Activity of CD20-Specific IgG Correlates With Bivalent Antigen Binding and C1q Binding Strength

“…A general fast association for C1q is in agreement with previous reports and also explains the fast onset of CDC, reaching maximum killing levels within 10 min even at C1q concentrations as low as 1 µg/ml (21,38). The apparent affinity for C1q binding to OFA coated Daudi cells, as determined with an end-point assay, has been reported previously to be 16 nM (21,38,59), which is close to the value reported here for the less stable interaction component. Data resolution for end-point affinity measurements is typically not sufficient to discriminate between individual interaction components with different affinities.…”

Special Issue: The Role of Complement in Cancer Immunotherapy

“…Although the efficacy of rituximab was and is clearly demonstrable, its apparent mechanisms of action were the subject of considerable controversy; however, its putative "apoptotic induction" has been set aside, and its therapeutic action has been clearly demonstrated to require immune effector mechanisms, which include complement-dependent cytotoxicity (CDC) [2,3]. Moreover, the basic science studies of rituximab have led to findings that have been most immediately applicable to understanding how other tumor-specific mAbs function, and the outcome of the studies of rituximab have also set the stage for the development of much more effective second-and third-generation mAbs that target CD20 as well as other tumor-associated antigens [4].…”

“…Golay and Taylor also cite several bedside to bench investigations which indicate that upregulation of complement control proteins is not a mechanism of resistance employed by CLL cells to inhibit CDC mediated by rituximab or ofatumumab. Finally, both reviews discuss the important advance in the design of more effective complement-activating mAbs based on generating IgG molecules that more readily form hexamers upon binding to cells, thus allowing for more effective chelation of C1q [4]. The illustrations in both of these articles are elegant and informative.…”

“…The review by Taylor and Lindorfer examines in detail key steps in complement mediated lysis of B cells that are opsonized with highly effective complement-activating mAbs (hexamer-forming) specific for CD20 and CD37 [4]. There is indeed considerable colocalization of cell-bound mAb with C1q, and this is rapidly followed by "nearby" covalent deposition of C3b (colocalized with bound mAb), which is soon followed by assembly and deposition of the membrane attack complex (MAC, C5b-9) of complement.…”

“…Whether CDC can be accomplished in the absence of C9 for these yet-to-be-developed mAbs is not clear, but based on the observations with the traditionally resistant CLL cells it is likely that C9 will not be required. Another lesson learned from these studies is that the action of mAbs that are particularly effective at activating complement exceeds the molecular thresholds for C1q binding and C3b deposition required for the generation of large quantities of the MAC, and therefore these mAbs are capable of overwhelming the natural defenses (including complement control proteins) expressed by tumor cells [4].…”