Emergence of Fc-Gamma-Riib-Dominance Contributes to Resistance to Therapeutic Antibodies in Patients with Chronic Lymphocytic Leukaemia

Aim:Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia. Whilst therapeutic antibodies show clinical activity in CLL patients, resistance develops. Thus, identifying mechanisms of antibody resistance and methods to reduce resistance would be valuable in managing CLL.

Results:In this study we show that a therapeutic antibody against CD62L is able to induce antibody-dependent cell mediated cytotoxicity (ADCC) and phagocytosis (ADP) in primary cultures of CLL cells. Significantly, we observed that patients with stable disease retained sensitivity to CD62L-Ab whilst untreated patients, whose disease progressed, became progressively resistant to CD62L-Ab. Using strategies to enrich for monocytes we were able to show that the CD62L-Ab dependent killing was attributable to an FcγR-dependent mechanism within the monocyte derived cell (MDC) fraction of PBMCs. Transcriptomic profiling and marker analysis indicated that the MDCs acquired a macrophage phenotype. Both MDCs from antibody-sensitive or antibody-resistant patients were able to bind Ab-bound CLL cells equally. Moreover, resistance could not be attributed to reduced numbers of monocytes or macrophages or to distinct subtypes of monocytes or macrophages. Using pharmacological inhibitors of the activating pathway of FcγR signaling and the inhibitory FcγRIIB pathway we were able to show that the antibody resistance in MDCs, derived from patients with CLL, was due to the emerging dominance of the FcγRIIb pathway relative to the activating FcγR pathways.

We examined whether the differential sensitivity to CD62L-Ab was also evident for anti-CD20 antibodies used clinically for CLL. Rituximab showed only moderate activity in vitro and no clear difference in cytotoxicity was observed between patients who were previously identified as being resistant or sensitive to the CD62L antibody. Obinutuzumab invoked similar differential cell killing in PBMCs from patients sensitive to, or resistant to, CD62L-Ab. Further comparison indicated that CD62L-Ab and obinutuzumab induced similar malignant B cell binding to MDCs and ADP in contrast to rituximab. Finally, similar to anti-CD62L, ADCC/ADP response to obinutuzumab was reduced following treatment  of sensitive cultures with a syk or BTK inhibitor and increased in MDCs derived from resistant patients treated with a Ship1 inhibitor.

Conclusions: These data establish, for the first time, that MDCs derived from CLL patients may switch from an antibody sensitive phenotype to an antibody-resistant phenotype as disease progresses.  Significantly, we show that the resistance to MDC-mediated ADCC/ADP may be reversed by the inhibition of FcγRIIB with pharmacological modifiers.