Development And Characterization Of High-affinity Anti-CD20 Monoclonal Antibodies

CD20 is a cell-surface marker expressed on mature B cells and most malignant B cells, but not stem or plasma cells. It is an ideal target for monoclonal antibodies (mAb), such as rituximab, as it is expressed at high levels on most B-cell malignancies, but does not become internalized or shed from the plasma membrane following mAb treatment. This allows the antibody to persist on the cell surface for extended periods to deliver sustained immunological attack from complement and FcR expressing cells.The chimeric anti-CD20 mAb rituximab was approved for use in relapsed or refractory low-grade or follicular B-cell non-Hodgkin's lymphoma (NHL) in 1997. Previous studies have suggested that several main mechanisms might be involved in providing therapeutic efficacy, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Despite the effectiveness of rituximab, only 48% of patients respond to the treatment and complete responses are less than 10%. In addition, a significant number of patients have progressive disease during antibody therapy. There is an urgent need to develop more effective anti-CD20 antibodies to further improve the efficacy of antibody therapy for B-cell lymphomas. In our previous study, we developed the crystal structure of rituximab-Fab in complex with the CD20 epitope peptide. Based on this crystal structure, we used computer-aided design to generate three single-point mutants with higher affinity (Rituximab_D57R,Rituximab_Y102R,Rituximab_Y102S). Our data showed that these high-affinity mutants displayed similar CDC activity to that of rituximab, but their ADCC activity was greater than that of rituximab.In our previous study, a humanized anti-CD20 antibody, hu8E4, was generated by complementarity-determing region grafting method. Hu8E4 was as effective as rituximab in mediating antibody-dependent cellular cytotoxicity in B-lymphoma cells, but it exhibited much more potent complement-dependent cytotoxicity than rituximab. In our present study, we attempted to enhance the affinity of hu8E4 in order to further improve its anti-tumor activity. To employ the computer-aided design to enhance antibody binding affinity, we need to acquire the crystal structure. However, the crystal structure of antigen-antibody complexes was diffcult to obtain. Previous studies have suggested that most of anti-CD20 antibodies which employed by the traditional hybridoma technology compared with rituximab with the same epitope peptide. So we speculated whether the affinity-improving mutations of an antibody could be transplanted into the corresponding places of another antibody to improve its affinity when these two antibodies had similar eiptopes. In our previous study, we employed the phage display peptide libraries technology to select peptides binding to hu8E4. We found that hu8E4 and rituximab recognize the same epitope peptide. So we try to transplant the three affinity-improving mutations (D57R,Y102R,Y102S) to hu8E4 in order to enhance antibody binding affinity. In addition to verify its effectiveness, we also transplant the three affinity-improving mutations (D57R,Y102R,Y102S) to chimeric antibody 2H7 which also compared with rituximab with the same epitope peptide.The results showed that the avidity of hu8E4D57R and hu8E4_Y102S was higher than that of hu8E4, while the avidity of hu8E4_Y102S was lower than that of hu8E4. The three mutants (hu8E4_D57R,hu8E4_Y102R,hu8E4_Y102S) were as effective as hu8E4 in CDC, while they were more effective in mediating ADCC in an affinity-dependent manner. Our data indicated that the avidity of C2H7_D57R and C2H7_Y102R was higher than that of C2H7, while the avidity of C2H7_Y102S was lower than that of C2H7. The three mutants (C2H7D57R,C2H7_Y102R,C2H7_Y102S) were as effective as C2H7 in CDC, while they were more effective in mediating ADCC in an affinity-dependent manner.Here we have obtained the high-affinity mutants of the humanized anti-CD20 monoclonal antibody, hu8E4. The high-affinity mutants showed more potent anti-tumor activity than that of the parental antibody. Our study suggested that the affinity-improving mutations of an antibody could be transplanted into the corresponding places of another antibody to improve its affinity when these two antibodies had similar eiptopes. These high-affinity mutants of hu8E4 might be the promising therapeutic agents for the treatment of B-cell lymphomas.