Development of novel humanized anti‐CD20 antibodies based on affinity constant and epitope

Abstract

We describe novel humanized anti-CD20 monoclonal antibodies (mAbs) developed for therapeutic use on the basis of their physicochemical properties and cellular cytotoxicity. A distinct correlation between apparent dissociation constants (K d ) and apoptotic activity for eight murine anti-CD20 mAbs (OUBM1-OUBM8) and previously-developed murine anti-CD20 mAbs enabled us to categorize anti-CD20 mAbs into two groups. Group A mAbs had lower K d values and did not induce definite apoptosis, while Group B mAbs had greater K d values and did induce definite apoptosis. A murine version mAb of rituximab, 2B8, belongs to Group B. An epitope analysis showed that the epitope of two murine mAbs, OUBM3 and OUBM6, differed from that of 2B8 or 2F2 (ofatumumab). Two mAbs, OUBM3 from Group A and OUBM6 from Group B, were selected and humanized. As expected, the humanized OUBM3 with the lower K d did not induce apoptosis, while the humanized OUBM6 (hOUBM6) with the greater K d did. Both hOU-BM3 and hOUBM6 induced highly-effective, complement-dependent cytotoxicity and antibody-dependent, cell-mediated cytotoxicity against Burkitt's and follicular lymphomas. Importantly, hOUBM6 exhibited cellular cytotoxicity against diffuse, large B cells that are less effectively depleted by rituximab and also exhibited effective cytotoxicity against tumor cells from human CD20(+) leukemia and lymphoma patients. These results suggest the potential impact of the further development of our anti-CD20 mAbs. Our study shows that the selection of mAbs based on their physicochemical parameters, followed by the biological activity assessment for the selected mAbs, is a rational and efficient approach for pharmaceutical mAb development. (Cancer Sci 2010; 101: 201-209) R ecently, the application of immunotherapy using monoclonal antibodies (mAbs) has increased considerably, and numerous mAb candidates are under development or are being assessed in clinical trials. In the case of mAb development using murine mAbs, the degree of complement-dependent cytotoxicity (CDC) and antibody-dependent, cell-mediated cytotoxicity (ADCC) activity can be measured after humanization, which implies that it is difficult to evaluate the clinical effectiveness of these antibodies (Abs) before humanization. The humanization of large numbers of murine mAbs without data to support future clinical use requires massive investments in terms of time, labor, and funding. Furthermore, the evaluation of bioactivity is often dependent on the assay systems themselves. Therefore, it is highly desirable to select candidates for humanization on the basis of the intrinsic parameters of the mAb itself, such as its dissociation constant and epitope, at an early stage of mAb development. In order to establish an efficient and rational method of developing mAbs, we focused on anti-CD20 mAbs as the ideal study target for studying the intrinsic properties of mAbs, because data on these Abs is available.CD20 is a B-cell-specific tetra-transmembrane protein with a molecular weight of 33-37 kDa. It i...

Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties

“…In addition to the difference in binding sites between ofatumumab and rituximab, studies have suggested that ofatumumab dissociates more slowly from the cell surface than rituximab 35 and exhibits greater CDC activity than rituximab in various B cell lines. 35,48,75,77 Furthermore, CDC by ofatumumab was found to be less dependent on the cell-surface density of CD20 than CDC by rituximab. 48 The differential action of ofatumumab on the complement has been supported by direct visualization of complement-mediated cell killing obtained using spinningdisk confocal microscopy.…”

“…83 In preclinical studies, variants of hOUBM6 showed higher CDC levels, similar or higher ADCC levels and similar depletion of leukemia and lymphoma cells compared with rituximab. 75 Residues A170 and P172 of CD20 are not essential for binding of hOUBM3 and hOUBM6, suggesting that the epitope for these antibodies indeed differs from that of rituximab. According to the limited available data, the epitope for hOUBM6 includes the motifs 287 75 Researchers reporting preclinical studies of a series of hOUBM3 and hOUBM6 variants recently proposed a classification scheme based on the affinity (measured by the dissociation constant) and the epitope of antibodies, rather than biological effects as used to categorize Type I and II anti-CD20 antibodies.…”

“…75 Residues A170 and P172 of CD20 are not essential for binding of hOUBM3 and hOUBM6, suggesting that the epitope for these antibodies indeed differs from that of rituximab. According to the limited available data, the epitope for hOUBM6 includes the motifs 287 75 Researchers reporting preclinical studies of a series of hOUBM3 and hOUBM6 variants recently proposed a classification scheme based on the affinity (measured by the dissociation constant) and the epitope of antibodies, rather than biological effects as used to categorize Type I and II anti-CD20 antibodies. 75 The affinity was correlated with potential to induce direct cell death, allowing antibodies to be defined into Group A and Group B antibodies.…”

“…According to the limited available data, the epitope for hOUBM6 includes the motifs 287 75 Researchers reporting preclinical studies of a series of hOUBM3 and hOUBM6 variants recently proposed a classification scheme based on the affinity (measured by the dissociation constant) and the epitope of antibodies, rather than biological effects as used to categorize Type I and II anti-CD20 antibodies. 75 The affinity was correlated with potential to induce direct cell death, allowing antibodies to be defined into Group A and Group B antibodies. Group A antibodies (hOUBM3, hOUBM6 clones with lower K d and ofatumumab) exhibited high affinity and did not induce direct cell death in lymphoma cells.…”

“…20,21 Ofatumumab is being studied in patients with lymphomas either as a single agent or in combination with chemotherapy. 58,[72][73][74] Like rituximab, ofatumumab shows Type I anti-CD20 activity, including CD20 rafting and CDC activity, 35,75 but binding studies suggest that ofatumumab recognizes an epitope different from that of rituximab. While the binding of rituximab is prevented by mutation of the A170/P172 residues, site-directed mutagenesis has shown that such mutations in the large extracellular loop of CD20 do not affect the binding of ofatumumab.…”