Rational Design Of Novel CD20 Antibodies And Their Biological Function Study

lymphocytes in early stages of differentiation and by most B-cell lymphomas. CD20 is an ideal target for monoclonal antibodies (mAbs), as it is expressed at high levels on most B-cell malignancies but does not become internalized or shed from the plasma membrane after mAb treatment. The mouse/human chimeric anti-CD20 antibody, Rituximab, is the first therapeutic mAb approved for the treatment of relapsed/refractory low-grade or follicular B-cell non-Hodgkin lymphomas. Previous studies have suggested that several mechanisms might be involved in providing therapeutic efficacy, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and the induction of apoptosis. The relative contributions of these different mechanisms of action are still a matter of debate. Despite widespread use of the anti-CD20 monoclonal antibody (mAb), Rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. A better understanding of Rituximab-mediated killing mechanisms is essential to develop more effective therapeutic agents. In this study, we modulated the binding property of Rituximab by introducing several point mutations in its complementarity-determining regions. The data showed that changing the binding avidity of Rituximab in the range from 10-8 to 10-10 M could regulate its antibody-dependent cellular cytotoxicity but not affect its complement-dependent cytotoxicity and apoptosis-inducing activity in B-lymphoma cells. Contradictory to previous findings, we found that the complement-dependent cytotoxicity potency of CD20 mAb was independent of the off-rate. Despite still being a type I CD20 mAb, a Rituximab triple mutant (H57DE/H102YK/L93NR), which had a similar binding avidity to a double mutant (H57DE/H102YK), was unexpectedly found to have extremely potent apoptosis-inducing activity. Moreover, this triple mutant, which was demonstrated to efficiently initiate both caspase-dependent and -independent apoptosis, exhibited potent in vivo therapeutic efficacy, even in the Rituximab resistant lymphoma model, suggesting that it might be a promising therapeutic agent for B-cell lymphomas.