Background In patients with Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE), who have poor responses to standard doses of rituximab (RTX), enhanced B-cell depletion and improved clinical responses can be achieved with higher doses of RTX. Defects in complement, NK cell function and CD11b polymorphisms associated with SLE may impair the effector mechanisms evoked by RTX whilst high BAFF levels may confer B cells some resistance to apoptosis. Obinutuzumab (OBZ, a Type II anti-CD20 monoclonal antibody [mAb] with afucosylated Fc portion and enhanced affinity for Fcγ receptor III) is more efficient than Rituximab (RTX, a Type I mAb) at inducing malignant B cell cytotoxicity and has recently entered Phase II clinical trials in SLE.
Objectives To compare the effects of RTX and OBZ against B cells from patients with RA and SLE in vitro and dissect any differences between the two mAbs in evoking effector mechanisms in RA and SLE.
Methods We used flow cytometry to perform: in vitro whole blood B cell depletion assays to compare the efficiency of OBZ and RTX in depleting target B cells; surface fluorescence-quenching assays to determine the extent of mAb internalization; complement dependent cellular cytotoxicity (CDC) assays to assess the ability to recruit complement and lyse target cells; NK cell degranulation assays assessing surface expression of CD107a as a measure of antibody-dependent cellular cytotoxicity (ADCC); and neutrophil activation assays assessing the surface expression of CD62L and CD11B. Annexin V positivity represented cell death in direct cell death (DCD) assay. OBZ-gly (OBZ with Fc glycosylation similar to RTX) was used to assess the effects of afucosylation. All mAb were used at 1μg/mL except for the CDC and DCD assays (10μg/mL).
Results OBZ was >2 fold more efficient than RTX at inducing cytotoxicity in B cells from patients with RA (n=31) and SLE (n=34) in whole blood assays. It was also internalized by isolated B cells from SLE patients (n=6) to a significantly lower extent than RTX with a median surface accessible mAb of 75% and 59%, respectively, after 6 hours of incubation. OBZ was at least twice as efficient at inducing NK cell activation, in RA (n=13) and SLE (n=19); and also activated neutrophils more efficiently than RTX in SLE (n=15) (Figure 3). In contrast, RTX was significantly more efficient than OBZ at evoking CDC of isolated B cells (SLE, n=6). OBZ was also more efficient at inducing direct cell death in CD19+ B-cells as a whole and in naïve (IgD+CD27-); and switched (IgD-CD27+) memory B-cells, a higher frequency of which is associated with poor clinical response after RTX.
Conclusions Obinutuzumab is more efficient than rituximab at inducing B cell cytotoxicity in vitro in both RA and SLE samples. Enhanced direct cell death, reduced CD20 internalization and superior FcR-mediated effector mechanisms are observed, while complement mediated cytotoxicity is reduced. These data provide a mechanistic basis for considering Obinutuzumab as an alternative B cell depleting agent in RA and SLE, in particular.
Disclosure of Interest V. Reddy: None declared, C. Klein Employee of: Roche Pharmaceuticals, D. Isenberg Speakers bureau: consultation fees paid directly into a local charity, G. Cambridge: None declared, M. Cragg Grant/research support from: research grant from Roche, M. Leandro Speakers bureau: travel support to attend conferences