Type II anti-CD20 mAbs depleted B cells more effectively than type I mAbs via a complement and apoptosis-independent mechanism that requires Fc/FcR engagement. (A) Systemic depletion: hCD20 Tg mice (BALB/c background) received 250 μg anti-CD20 mAb or isotype matched control intravenously on day 0 and the number of circulating B cells assessed. Points indicate means; n ≥ 4 mice for each time point from 2 to 4 independent experiments. (B) Adoptive transfer: hCD20 Tg target (T) or WT nontarget (NT) BALB/c splenocytes labeled with high or low CFSE, respectively, were injected intravenously into BALB/c mice. Twenty-four hours later, mice received mAbs (1 μg, intravenously); and 16 hours later spleens analyzed to determine the T/NT ratio. Left panel: bars represent mean ± SD; n = 3 to 5 mice for each treatment group. *Both type II mAbs significantly differ from both type I mAb: Rit m2a versus FGM6 (P < .02); Rit m2a versus Tosit P < .002; 1F5 versus FGM6 and 1F5 versus Tosit (P < .005). Right panel: Typical dot-plot data. (C) Dose-response to Rit m2a and tositumomab (Tosit) in a similar adoptive transfer: bars represent mean and range; n = 2 mice at each concentration. (D) Contribution of effector mechanisms: Ctl, transfer of T and NT into WT recipients as in panel B; CDC, NT, and T cells transferred into complement-deficient (C1q−/− and C3−/−) mice; programmed cell death, hCD20 Tg and hCD20 × Vav-Bcl-2 double Tg (both T), and WT (NT) labeled with high CFSE, low CFSE, and PKH26, respectively, transferred into WT mice and the level of both T compared with the NT; Fc/FcγR, T, and NT cells transferred into γ−/− or clodronate-treated (Clod) WT mice; also shown is the activity of F(ab′)2 fragments in WT recipients. Bars represent mean ± SD; n = 3 mice for each treatment group. Each condition is representative of at least 2 independent experiments. (E) Poor type I depletion is not the result of shaving: Transfer of T and NT cells into WT and CD64−/− mice as in panel B with Ctl, Rit m2a, or Tosit mAb. Bars represent mean and range; n = 2 mice for each treatment group; representative of at least 2 independent experiments.
