Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose
Incomplete B-cell depletion using rituximab (RTX) is associated with poor clinical response in some individuals with RA and SLE, in particular. However, the precise mechanisms of resistance to depletion with RTX (type1 anti-CD20 monoclonal antibodies, mAbs) are poorly understood. Improving depletion may augment clinical response. Newer mAbs such as GA101 (type2) are more potent than RTX at deleting malignant B cells.
Methods
We included 5 healthy controls (HC), 15 patients with RA and 16 with SLE. An in vitro autologous whole blood depletion assay was used to compare BHH2 (type2, glycosylated GA101) with RTX (type1) and B cell lysing potential of mAbs was defined as cytotoxicity index (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 analysed by flow cytometry for CD45+ lymphocytes, CD3+ Tcells and CD19+ Bcells. The CTI was calculated using the formula:CTI of mAb=100-[(number of B:Tcells in sample without mAb-number of B:Tcells with mAb)/number of B:T cells in sample without mAb) X 100]. 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. 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. Prior incubation with AT10 (a mAb directed against Fcgamma receptor II, CD32) significantly reduced internalisation of type1 mAbs to a greater extent than type 2 mAbs, a mean reduction of 12% vs 4%, respectively (p<0.005). Further, a significant variability was noted between patients in the extent to which internalisation of RTX, but not BHH2, was reduced by AT10.
Conclusion
Type 2 anti-CD20 antibodies are more efficient than type1 (rituximab) at lysing B cells from patients with RA and SLE. 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. Fcgamma receptor IIb facilitates rapid internalisation of rituximab, but not type2 mAbs, by B cells from patients with RA and SLE, which may contribute to its inferior ability to lyse B cells. This study provides a mechanistic basis for considering type2 mAbs for clinical use in RA and SLE.
Disclosure:
V. Reddy,
None;
G. Cambridge,
None;
D. A. Isenberg,
None;
M. Cragg,
None;
M. J. Leandro,
None.
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