The Bs20x22 anti-CD20-CD22 bispecific antibody has more lymphomacidal activity than do the parent antibodies alone

Abstract

Previous studies have shown that bispecific antibodies that target both CD20 and CD22 have in vivo lymphomacidal properties. We developed a CD20-CD22 bispecific antibody (Bs20x22) from anti-CD20 and the anti-CD22 monoclonal antibodies (mAb), rituximab and HB22.7, respectively. Bs20x22 was constructed using standard methods and was shown to specifically bind CD20 and CD22. In vitro cytotoxicity assays showed that Bs20x22 was three times more effective than either parent mAb alone and twice as effective as a combination of both parent mAb used at equimolar concentrations. Bs20x22 was also nearly four times more effective at inducing apoptosis than either mAb alone. Examination of the MAPK and SAPK signaling cascades revealed that Bs20x22 induced significantly more p38 phosphorylation than either mAb alone. In an in vivo human NHL xenograft model, treatment with Bs20x22 resulted in significantly greater tumor shrinkage and improved overall survival when compared to either mAb alone or treatment with a combination of HB22.7 and rituximab. The effect of the initial tumor volume was assessed by comparing the efficacy of Bs20x22 administered before xenografts grew versus treatment of established tumors; significantly, greater efficacy was found when treatment was initiated before tumors could become established.

Fig. 1

Transfected 293T or Ramos cells were stained with either rituximab, HB22.7, or Bs20x22, then washed and stained with the appropriate anti-human or anti-mouse fluorescent mAb. a CD20-transfected 293T cells + rituximab. b CD20-transfected 293T cells + Bs20x22. c CD22-transfected 293T cells + HB22.7. d CD22-transfected 293T cells + Bs20x22. e Ramos + Bs20x22 + anti-human Ig-TexasRed. f Ramos + Bs20x22 + anti-mouse Ig-FITC

Fig. 2

Raji cells were treated with rituximab (filled triangle), HB22.7 (empty circle), combination rituximab/HB22.7 (empty square), or Bs20x22 (filled circle) and assessed for cell viability (a). Raji and Ramos cells were assessed for apoptotic induction (b). Error bars represent the standard deviation *P value < 0.02 (Bs20x22 vs rituximab/HB22.7). **P value < 0.01 (Bs20x22 vs rituximab/HB22.7). ***P value < 0.01 (Bs20x22 vs all groups)

Fig. 3

Ramos cells were treated with rituximab, HB22.7, or two different preparations (labeled 1 and 2) of Bs20x22. MAP kinase activation (p-38, JNK, ERK1/2) was analyzed by Western blot. Anti-IgM was included as a positive control. This represents a consistent and representative experiment, which was done in duplicate

Fig. 4

Mice bearing Raji NHL xenografts were treated with rituximab (empty square), HB22.7 (empty triangle), combination rituximab/HB22.7 (solid square), Bs20x22 (filled diamond), or PBS control (empty diamond). Mice were assessed twice weekly for tumor volume (a) and survival (b). Mice bearing Raji NHL xenografts were treated with Bs20x22 before (“pre-emptive”, filled triangle) or after (“established”, filled circle) tumors were established and compared with untreated controls (filled squares) (c). Replicate experiments were done with consistent results with the data presented representing an analysis of all mice studied. A total of 16 mice pre-treatment group were used with the error bars representing the standard deviation