Response: Superior B cell–depleting activity of type II anti-CD20 mAb is not due to activation of complement

Our paper focuses on reporting that tositumomab (a type II anti–human CD20) greatly outperforms rituximab and other type I anti-CD20 monoclonal antibody (mAb) in depleting human CD20-expressing B cells in a transgenic mouse. This, despite the fact that the mAb were all of the same mouse IgG2a isotype, selected for maximum effector function, and recognized the same cross-blocking epitope on CD20.¹ Previous data in xenograft models has suggested that a type II mAb might have higher efficacy,² but our paper is the first to demonstrate this comprehensive difference in a fully syngeneic system using unmanipulated target cells in vivo.

The main effector difference between type I and II CD20 mAb is that type I, which translocate CD20 to lipid microdomains, activate complement very efficiently, whereas type II do not.³ Extensive investigations in previous work^2–4 and the current paper⁵ show that rituximab is at least 25 times more active at evoking complement-dependent cytotoxicity (CDC) than tositumomab (B1) in the presence of rat or human serum. The relatively low activity of tositumomab is confirmed by the data shown by Beurskens et al against highly sensitive targets under optimal conditions. This low activity prevents type II mAb, such as tositumomab, from mediating effective CDC against B-cell targets that are protected by complement regulatory proteins, such as CD55 and CD59.^6,7 Taken together, it is clear that if complement is an important effector mechanism, as we^2,3,8 and many others^9–13 have shown, it does not explain why tositumomab is so much more effective in the huCD20-transgenic mice. One potential explanation is that, in certain settings, complement is actually deleterious for therapy as recently suggested.¹⁴ To test this idea, we transferred huCD20-transgenic B cells into C1q-deficient mice and made the K322A mutant of Ritm2a (and 1F5; a second type I mAb), a substitution which is known to significantly reduce C1q recruitment in mouse IgG2 antibodies.^15,16 In answer to the question raised by Beurskens et al regarding the effectiveness of the K322A mutation in ablating CDC, we agree that the case for human IgG1 is unclear.¹⁷ In fact, the paper they cite^18(p4178) states, “Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1′.” Thus, we do not agree that the K322A substitution in mouse mAb is not removing CDC and our results in Figure 4A of our article clearly support this conclusion. Furthermore, this is an ideal modification for our work as it does not interfere with other effector functions, such as IgG binding to FcγR and FcRn.

Regardless of whether this mutation ablated or partially removed CDC activity, our results show that it made little difference to the efficacy of Ritm2a and did not convert its performance into that of a type II mAb. Therefore, we conclude that in this model, complement neither helps nor hinders efficacy of CD20 mAb and does not explain the outstanding efficacy of the type II reagent. We are currently exploring alternative mechanisms that explain such activity.

Authorship

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Prof Martin J. Glennie, Tenovus Research Laboratory, Cancer Sciences Division, University of Southampton, Southampton, United Kingdom SO16 6YD; e-mail: mjg@soton.ac.uk.