Abstract
Cellular activity of BM-ca, a novel humanized anti-CD20 antibody, was quantitatively compared with that of two other anti-CD20 antibodies used for clinical practice, rituximab and ofatumumab. The results of a complement-dependent cytotoxicity (CDC) assay revealed that the strongest antibody was ofatumumab, followed by BM-ca, with rituximab being the weakest. Ofatumumab and BM-ca were effective not only against rituximab-sensitive SU-DHL-4 cells but also against rituximab-resistant RC-K8 cells. In an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, although the effective concentrations against SU-DHL-4 cells were almost the same among these three antibodies, the maximum cytotoxic level was the highest for BM-ca. In an anti-cell proliferation assay using SU-DHL-4 cells, BM-ca was the most effective and ofatumumab, the weakest. Against RC-K8 cells, only BM-ca was effective. When combined with each of four cancer chemotherapeutics (prednisolone, vincristine, hydroxydaunorubicin, and cisplatin), BM-ca exerted the most effective combinatorial anti-cell proliferation activity. To assess the in vivo effect of BM-ca, we intravenously administered BM-ca into cynomolgus monkeys and found that the peripheral B-cell levels did not decrease in half of the animals. Sequencing of cDNA encoding CD20 of cynomolgus monkeys revealed that the responders and nonresponders had Leu/Pro (hetero) and Leu/Leu (homo) at amino acid (a.a.) position 160, respectively, suggesting that the epitope recognized by BM-ca was around this a.a. By analyzing reactivity to synthetic peptides, the epitope recognized by BM-ca was estimated to be a.a.'s 156–166, not shared with rituximab and ofatumumab. These results suggest BM-ca to be a promising anti-CD20 antibody having superior properties and recognizing a unique epitope.
Highlights
CD20 is a promising target molecule of antibody drugs for treating a variety of B-cell-related diseases, including B-cell lymphoma, B-cell chronic lymphocytic leukemia, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, etc [1]
There existed no significant differences in antibody-dependent cell-mediated cytotoxicity (ADCC) activities in RC-K8 cells, in SU-DHL-4 cells BM-ca lysed a larger number of target cells than the two other antibodies above plateau concentrations
Epitope analysis of BM-ca with synthetic peptides. Since these results strongly suggest that the region in the large extracellular loop around position 160 was the epitope recognized by BM-ca, we synthesized peptides of a.a. 140–166 of human (CD20_140-166H) and monkey (CD20_140-166H_A157V) sequences having Pro at position 160 and examined their ability to bind to BM-ca, rituximab, and ofatumumab
Summary
CD20 is a promising target molecule of antibody drugs for treating a variety of B-cell-related diseases, including B-cell lymphoma, B-cell chronic lymphocytic leukemia, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, etc [1]. Over 15 years have passed since the debut of rituximab, but up to now only a single anti-CD20 antibody, ofatumumab, has been approved, and it for only a single indication, chronic lymphocytic leukemia [5]. Many anti-CD20 antibodies under development have characteristics different from those of rituximab, for example, potentiated antibody-dependent cell-mediated cytotoxicity (ADCC) [6], stronger complement-dependent cytotoxicity (CDC) [7], no chimeric structure but rather a humanized or fully human structure, recognition of different epitopes, etc [1]. Such superiorities in vitro do not always reflect clinical efficacy. Even we do not sufficiently understand the key properties that allow these newer anti-CD20 antibodies to be more effective in vivo than the current champion, that is, rituximab
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