In vivo depletion using human and mouse type I and II antibodies. (A) Left panels show that transgenic hCD20 mice were administered 250 µg anti-CD20 by tail-vein injection and the percentage of circulating B220, CD19+ B cells measured over 90 days (n = 4). Right panels show statistical comparison of human vs mouse isotype mAbs at days 28 and 40. Statistical analyses were carried out using 2-way ANOVA with multiple comparisons and significance was accepted at ****P < .0001, ***P < .001, and **P < .01. (B) SPR analysis of anti-human CD20 mAbs (hIgG1, mIgG1, and mIgG2a) binding to mouse FcγRI, IIb, III, and IV. Recombinant, soluble FcγR proteins were passed over mAbs immobilized at 2000 RU. Sensorgrams are shown. (C) Binding comparison of type I and II anti-CD20 mAbs to transgenic hCD20 B cells. Cells were opsonized with 0.001 to 100 µg/mL anti-CD20 mAbs and analyzed by flow cytometry. The mean number of phycoerythrin (PE) molecules per cell was quantified by indirect staining with anti-mouse or anti-human Fc PE-conjugated F(ab′)2 and comparison of the geometric mean fluorescent index with BD QuantiBRITE beads. (D) The concentration of anti-CD20 mAbs in the sera of mice administered 250 µg of human IgG1 mAbs was determined by enzyme-linked immunosorbent assay; n = 4 mice per group. ND, not detectable. Bars represent mean ± standard deviation. Irr, WR17; OFA, ofatumumab; RTX, rituximab.
