Construction Of Novel Engineered Tetravalent Anti-CD20 Antibodies And Anti-tumor Activity

The mouse/human chimeric anti-CD20 antibody C2B8 (Rituximab) is the first therapeutic monoclonal antibody (mAb) approved for the treatment of relapsed/ refractory low-grade or follicular B-cell non-Hodgkin's lymphomas. Despite the effectiveness of C2B8, only 52% of patients respond when given 375 mg/m~2 weekly for 4 weeks. It has been shown that C2B8 is not effective in killing the lymphoma cells expressing low levels of CD20.In this study, two recombinant tetravalent chimeric anti-CD20 mAbs were generated using a procedure recommended for the preparation of bispecific antibodies. The secreted antibodies were purified from the culture supematants by affinity chromatography and tested for following activities: 1. To mediate CDC and to bind human complement component C1q; 2. To associate/dissociate to/from CD20- positive cells; 3. To mediate ADCC and to interact with Fc receptors (FcRs); 4. To evoke apoptosis and to deplete CD20-posifive cells in whole human blood; 5. To persist in the circulation of normal mice compared with their divalent antibody.In conclusion, the new TetraMAb mAbs presented here with longer half life are homogenous proteins with their divalent antibody but with higher functional affinities. Due to their increased valencies, these tetravalent mAb constructs are more cytotoxic to the CD20~+ tumor cells than their divalent counterpart and also have conserved effector function (they bind to C1q/FcRs) because of their human Fc. In addition, their extremely slow off-rates provide advantages for their use where they should function at low CD20-expressing tumor cells. We demonstrate that TetraMAbs induced apoptosis of CD20~+ cells in vitro and deplete these cells in whole human blood. The TetraMAbs with their binding to C1q and FcRs, mediating CDC and ADCC, inducing apoptosis, and depleting tumor cells in blood, hold considerable promise for improved clinical activity with their therapeutic activity in vivo.