Fc gamma receptor IIb on target B cells promotes rituximab internalization and reduces clinical efficacy

Abstract

The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heteroge- IntroductionThe anti-CD20 mAb rituximab has improved the overall survival of patients with follicular (FL) and diffuse large B-cell lymphoma (DLBCL). [1][2][3][4] However, in MCL, only modest responses are seen 5 and in CLL, fludarabine, cyclophosphamide and rituximab (FCR) therapy delivers improved responses but has yet to show a similar improvement in overall survival, 6 albeit the current follow-up is relatively short. Interestingly, those responses seen in CLL have often been achieved with high doses of rituximab, 6 suggesting that more mAb is needed to coat the targets or that it is consumed in some way. Even within rituximab-responsive lymphomas, a proportion of cases show resistance on first treatment with rituximab or eventually become resistant to rituximab-containing combination therapy (reviewed in Stolz et al 7 ). The molecular basis of this resistance and the observed sensitivity of different lymphoma subtypes is unclear (reviewed in Lim et al 8 ), but is highly relevant to improving outcomes.In addition to understanding target resistance, many groups are working to deliver anti-CD20 mAb reagents with improved affinity and more potent engagement of cytotoxic effectors. Anti-CD20 mAb can be defined as type I (eg, rituximab, ofatumumab) or type II (eg tositumumab, GA101), according to their ability to redistribute CD20 into lipid rafts in the plasma membrane and function in various effector assays. 9-11 It is still not clear what characteristics are required for the optimal reagent, but it is generally accepted that Fc:Fc ␥ receptor (Fc␥R) interactions are crucial to the efficacy of anti-CD20 mAb. [12][13][14][15] In particular, Fc␥RIIIa on myeloid effectors appears critical in controlling Ab potency and in keeping with this, lymphoma patients bearing the higher affinity 158V allele in Fc␥RIIIa respond better to rituximab compared with those with the low affinity 158F allotype, 16 leading many investigators to focus on augmenting the interaction of mAb with Fc␥RIIIa, for example via defucosylation. 17 Less attention has been given to the potential effects of the ITIM-containing inhibitory Fc␥R, Fc␥RIIb. Fc␥RIIb is a negative regulator of ITAM-containing receptors, such as the B-cell receptor (BCR) and the activatory Fc␥R. 18 Most hematopoietic cells coexpress inhibitory and activatory Fc␥R, and tumors are reported to be more sensitive to mAb immunotherapy in Fc␥RII Ϫ/Ϫ mice because of the removal of the inhibitory restraint of this receptor from myeloid effectors such as macro...

Antibody modulation: Limiting the efficacy of therapeutic antibodies

“…Furthermore, there was a negative correlation between the cell surface expression of FcγRIIB and the proportion of rituximab remaining on the cell surface after in vitro culture [19]. We also demonstrated that the ITIM of FcγRIIB was phosphorylated in response to rituximab, indicating that a direct interaction between the Fc domain of the mAb and the Fc-binding domain of the FcγR augmented internalisation.…”

“…By repeating our experiments under conditions in which direct cell-cell interaction was unlikely, Lim et al. demonstrated that a cis interaction between type I anti-CD20 mAb and FcγRIIB was required to augment internalisation [19], a process termed antibody bipolar bridging. Finally, we demonstrated reduced survival after rituximab treatment in patients with mantle cell lymphoma whose tumours expressed high levels of FcγRIIB, after treatment with rituximab-containing immunochemotherapy [19], compared to those expressing low levels.…”

“…The mechanism of internalisation of type I anti-CD20 mAb was investigated by Lim et al who demonstrated that the rate was slower in response to ligation by a F(ab') 2 fragment of rituximab, suggesting a potential role for Fc receptor engagement [19].…”

“…demonstrated that a cis interaction between type I anti-CD20 mAb and FcγRIIB was required to augment internalisation [19], a process termed antibody bipolar bridging. Finally, we demonstrated reduced survival after rituximab treatment in patients with mantle cell lymphoma whose tumours expressed high levels of FcγRIIB, after treatment with rituximab-containing immunochemotherapy [19], compared to those expressing low levels. This same observation was also made later in patients with follicular lymphoma treated with rituximab monotherapy [25], supporting the assertion that internalisation may reduce the therapeutic efficacy of type I anti-CD20 mAb therapy when used clinically.…”

“…In contrast, type II anti-CD20 mAb do not display either of these properties but instead evoke strong homotypic adhesion [14] and a non-apoptotic form of lysosomal cell death [14][15][16][17]. In addition, we observed that type I anti-CD20 mAb undergo more rapid internalization from the cell surface, in contrast to type II mAb [18][19][20]. Below we discuss the various ways in which the study of these two different types of mAb have elucidated a number of mechanisms of mAb resistance, before discussing how they may be overcome.…”