Fig. 6: In vivo B-cell depletion using the rituximab IgG1 μtp fusion mAb.

a Balb/C mice were administered 100 μg rituximab hIgG1 μtp constructs i.v. and peripheral serum was collected at 2 h and days 1, 2, 6, 14, and 21. The concentration of mAb in the serum was calculated by ELISA (n = 3). b FcRn binding was analysed by loading hIgG1 μtp constructs onto an FcRn affinity column at 1 mg/ml pH 5.5, and eluting using a pH gradient up to pH 8.8. c A 1:1 ratio of CFSE-labelled hCD20 Tg splenocytes (high) and wt splenocytes (low) were adoptively transferred into C57 BL/6 mice i.v. followed 24 h later by 25 μg rituximab hIgG1 mAb constructs i.p. After 24 h mice were sacrificed and splenocytes stained for B220. B-cell depletion was calculated using a T:NT ratio of CFSE high (T) to CFSE-low (NT) B cells in the spleen of treated mice (n = 5). d hCD20 Tg Balb/C mice were administered 100 μg rituximab hIgG1 mAb constructs i.v. on day 0. Circulating B-cell levels were monitored on days 1, 2, and 7 by peripheral blood collection using CD19/B220 flow cytometry staining. B-cell depletion is expressed as a % of B cells compared to pre-mAb administration (n = 5). e Spleen and inguinal lymph nodes were harvested on day 15, and B-cell depletion was assessed using CD19/B220 flow cytometry staining (n = 5). f Serum samples collected at each time point were used to determine circulating IgG concentration by ELISA (n = 5). Individual data points and mean are plotted from biologically independent animals. Statistical analysis was carried out by one-way ANOVA.