Complement Activation and Complement-Mediated Killing of B Cells Promoted by Anti-CD20 Monoclonal Antibodies (mAb) Rituximab and Ofatumumab Are Rapid, and Ofatumumab Kills Cells More Rapidly and with Greater Efficacy.
Authors: Taylor Ronald P. , Pawluczkowycz Andrew W. , Beum Paul V. , Lindorfer Margaret A. , Beurskens Frank , van de Winkel Jan , Parren Paul 
Published: April 2020

Abstract
We reacted four different CD20+ cell lines (ARH77, Daudi, Raji, and Z138) with either rituximab, ofatumumab or with no mAb, in 50% normal human serum (NHS) as a complement source. At 10 ug/ml, both mAbs bind to targeted cells quickly (t1/2 &lt; 2 min at 37°C), but there are substantial differences in the rates and efficacy at which rituximab and ofatumumab promote C3b deposition and subsequent complement-mediated cytotoxicity (CMC). After a 3 min incubation of Raji cells with rituximab, 55±3% (n=3, this and all experiments) of the cells were killed and deposited C3b corresponded to 123,000±5,000 molecules of equivalent soluble fluorochrome (MESF); comparable values for cells reacted with ofatumumab were 94±1% dead, and 173,000±19,000 MESF. Even after 12 min only 72±1% of the Raji cells had been killed by rituximab, and C3b deposition mediated respectively by rituximab and ofatumumab was 176,000±10,000 and 300,000±60,000 MESF. A similar experiment with Z138 cells led to the following results for CMC and for C3b deposition after 3 min: rituximab, 41±4% killing and 86,000±7000 MESF; ofatumumab, 83±1% killing and 275,000±25000 MESF. Although ARH77 cells are resistant to rituximab-mediated killing in NHS (26% dead after 60 min, compared to the no mAb background of 22%), these cells are killed rapidly by ofatumumab: 60–80% killing in &lt;15 min. We have reported that infusion of rituximab in patients with chronic lymphocytic leukemia (CLL) can lead to depletion of complement, likely a consequence of substantial complement activation and consumption mediated by the rituximab-CD20 immune complexes on the large numbers of circulating CLL cells. In the absence of effector cells in vitro killing of CD20+ targeted cells by both mAbs shows an absolute requirement for complement. Therefore, we evaluated the effect of target cell concentrations on CMC, to test the hypothesis that killing can be compromised due to exhaustion of complement even if the amount of mAb present is adequate to opsonize the cells. Indeed, whereas CMC of Z138 cells mediated by rituximab and ofatumumab gave 30% and 85% respective killing after one hour for cell concentrations between 1×106 and 1×107 cells/ml, killing was reduced to 15% and 30% when the cell concentrations were raised to 1 ×108 cells/ml. However, we found that killing could be restored simply by providing additional NHS as a complement source, with no requirement for additional mAb. In a similar experiment, Raji cells at 1 ×108 cells/ml were incubated for 15 min in 50% NHS with 10 ug/ml of either mAb; the cells were then diluted 10-fold by the addition of buffer or 50% NHS and incubated for another 15 min. CMC by rituximab corresponded to 26% (addition of buffer) and 45% (addition of NHS); comparable CMC for ofatumumab gave 49% and 82% killing. Translation of these findings to the clinic suggests that at high cell burdens, supplementation of a patient’s serum with a complement source may enhance mAb-mediated killing of CD20+ cells. This concept may be important in certain indications for optimizing the clinical efficacy of ofatumumab, which demonstrates a substantially higher level of complement activation and CMC of targeted cells than rituximab.