The Novel Bispecific Antibody [(CD20)₂xCD16] Efficiently Triggers Lysis of Neoplastic B Cells

Abstract 2846

Although the CD20 antibody rituximab considerably improved the treatment of B-cell neoplasias, novel optimized CD20-targeting strategies may still offer therapeutic gains. Analysis of Fc receptor (FcR) polymorphisms of rituximab-treated patients and animal studies demonstrated the crucial role for activating FcRs and effector cell recruitment. In the current study a novel CD20-directed bispecific antibody (bsab) [(CD20)₂×CD16] in the tribody format was evaluated to enhance antibody dependent cellular cytotoxicity (ADCC). The heterodimeric molecule forms a CD16-directed F(ab) fragment with a CD20-directed scFv c-terminally fused to both peptide chains. [(CD20)₂×CD16] was expressed in HEK-293T cells and purified using affinity chromatography. Specific binding to CD20- or FcγRIIIa- (CD16a) positive cells was demonstrated by flow cytometry. No significant difference in binding to both CD16a alloforms (CD16a-F158 and CD16a-V158) was observed. In chromium release assays [(CD20)₂×CD16] mediated specific lysis of different burkitt's lymphoma cell lines in a dose-dependent manner even at low picomolar concentrations. Potency of target cell lysis was improved compared to rituximab (10-fold) irrespective of the effector cell donors' CD16a V/F-polymorphism status. In addition, the bsab triggered lysis of primary tumor cells from 12/12 lymphoma patients (CLL; MCL; MZL). [(CD20)₂×CD16] triggered significantly stronger target cell lysis than rituximab or the parental CD20 antibody. Importantly, specific lysis of primary tumor cells was also observed using autologous NK cells as effector cells, suggesting that [(CD20)₂×CD16] is capable in triggering tumor cell lysis in the presence of a strong MHC I inhibitory signal. Interestingly, when B-cell depletion was analyzed in whole blood, significantly higher depletion rates were obtained with the bsab compared to rituximab (75% vs 25%). Depletion of B cells was accompanied by activation of NK cells, as evidenced by CD69 expression on NK cells. Finally, NOD-SCID mice were transplanted with CD34-positive cord blood cells to reconstitute a humanized hematopoietic system. Distribution and numbers of engrafted human B cells and human CD16-positive effector cells were analyzed by flow cytometry. CD16 was expressed on splenic monocytes and NK cells. After daily injection of [(CD20)₂×CD16] for three days a significant reduction of B cells was observed. Thus, the CD16-directed bispecific antibody [(CD20)₂×CD16] may extend the therapeutic range and represents a promising candidate for further clinical development.