Background A failure to achieve complete B-cell depletion using rituximab (RTX) may, at least in part, explain reduced efficacy noted in some individuals with Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE), in particular. However, there remains insufficient data on the precise mechanisms of resistance to rituximab-induced B cell cytotoxicity in RA and SLE. Newer anti-CD20 monoclonal antibodies (mAbs) such as GA101 (type2) have been shown to be superior to RTX (type1) in lysing B cells from patients with B cell malignancies. Therefore, we investigated the ability of type1 and type2 mAbs to lyse B cells in RA and SLE.
Methods We included 5 healthy controls (HC), 15 patients with RA and 16 with SLE. Whole blood depletion assay was performed in 5 HC, 13 with RA and 12 with SLE. We compared BHH2 (type2, glycosylated GA101) with RTX (type1). B cell lysing of mAbs was defined as cytotoxicity index (CTI). An in vitro autologous whole blood depletion assay was used to assess the CTI. Briefly, 100μl of heparinised blood was incubated with either RTX, BHH2 or without antibody, at a concentration of 1μg/ml at 37oC, 5% CO2for 24hours. Samples were then analysed by flow cytometry for CD45 (all lymphocytes), CD3 (T cells) and CD19 (B cells). The CTI was calculated using the formula:CTI of mAb=100-[(number of B:Tcells in sample without antibody-number of B:Tcells with mAb)/number of B:T cells in sample without antibody) X 100] and the mean from triplicate well calculated. Surface fluorescence quenching assay using isolated B cells was performed to assess for the internalisation of mAbs. Paired “t” test or Mann-Whitney U test was used to compare groups, as appropriate.
Results Whole Blood Depletion: BHH2 (type2 mAb) was significantly more efficient than RTX (type1 mAb) at lysing B cells, in vitro, in all groups. The mean ± SD CTI of BHH2 vs RTX in HC was 65±13 vs 45±24 (p=0.04); in RA, 61±12 vs 28±18 (p<0.0001); and in SLE, 38±17 vs 13±10 (p<0.0001). Thus, a hierarchy of B cell susceptibility to lysis by RTX was noted with HC > RA >SLE. Interestingly, BHH2 lyses the RA cells as well as controls - i.e. it fully overcomes the defect in RA whereas it doesn't in SLE.
Internalisation: We performed a surface fluorescence-quenching assay at 6 and 24 hours. At both time points, a significantly greater % of BHH2 was accessible on surface when compared with RTX, in all groups. The mean ± SD % surface accessible mAbs, after 6 hours of incubation for BHH2 vs RTX was 72±6 vs 57±11, 68±8 vs 57±12 (p<0.005) and 71±10 vs 63±12 (p<0.005) whereas after 24 hours of incubation it was 47±15 vs 28±12, 45±25 vs 36±15 and 59±9 vs 42±14 (p<0.005 for all), for HC, RA and SLE, respectively.
Conclusions Our data shows that type 2 anti-CD20 antibodies are more efficient than type1 (rituximab) at lysing B cells from patients with RA and SLE. Rituximab appears to be internalised by B cells from patients with RA and SLE, which may contribute to its inferior ability to lyse B cells. B cells from patients with SLE may be less susceptible to lysis in vitro by rituximab when compared with B cells from patients with RA and healthy individuals. This study provides a mechanistic basis for considering type2 mAbs for clinical use in RA and SLE.
Acknowledgements The work was supported by the Doris Hillier Grant and Arthritis Research UK.
Disclosure of Interest None declared