Furthermore, we compared their anti-cell proliferation activities in combination with cancer chemotherapeutic drugs

Furthermore, we compared their anti-cell proliferation activities in combination with cancer chemotherapeutic drugs. 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 156166, 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. Keywords:Anti-CD20 antibody, cancer chemotherapeutics, epitope, lymphoma, ofatumumab, rituximab == Introduction == 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, etc1. Rituximab, which is the top runner of anti-CD20 antibody drugs, was launched in 1997 in the United States (-)-Huperzine A and is now used worldwide for a variety of indications1,2. After the remarkable success of rituximab, many other attempts to develop other anti-CD20 antibodies were made3,4. Over 15 years have passed since the debut of rituximab, but up to now only a single anti-CD20 antibody, ofatumumab, has been (-)-Huperzine A approved, and it for only a single indication, chronic lymphocytic leukemia5. 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, etc1. However, such superiorities in vitro do not always reflect clinical efficacy. In other words, even now 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. BM-ca is a novel humanized anti-CD20 antibody having properties of both type-I and -II antibodies; that is, it has not only CDC but also direct cell death activities in addition to common ADCC activity8. BM-ca is now in a clinical phase-I (-)-Huperzine A study in Japan. In previous studies, some comparisons had been made with other anti-CD20 antibodies, but most of these studies were qualitative rather than quantitative8,9. In this study, we quantitatively compared the activity of BM-ca with that SCC3B of the two approved anti-CD20 antibodies, rituximab and ofatumumab, in vitro, that is, their CDC, ADCC, and direct anti-cell proliferation activities. Furthermore, we compared their anti-cell proliferation activities in combination with cancer chemotherapeutic drugs. To know the molecular basis of the differences among these anti-CD20 antibodies, the epitope on CD20 recognized by BM-ca was extensively characterized. In the course of this study, we became aware of the molecular heterogeneity of CD20 in the cynomolgus monkey (Macaca fascicularis), which heterogeneity significantly affected the binding of BM-ca, but not that of rituximab, to this monkey’s B cells. == Materials and Methods == == Cells and cell culture == Two human B-cell lymphoma cell lines, RC-K8 and SU-DHL-4, were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, (-)-Huperzine A Germany). Chinese hamster overy (CHO) cells constitutively expressing human CD20 molecules on their surface (CHO-CD20) were the same as those previously10. These cell lines were cultured in RPMI-1640 (Roswell Park Memorial Institute-1640) medium supplemented with 10% fetal bovine serum (FBS; for RC-K8 and SU-DHL-4) or in CHO-S-SFM II medium (Invitrogen, Carlsbad, CA) supplemented with 0.4 mg/mL G418 (for CHO-CD20) at 37C in a humidified chamber under a 5% CO2atmosphere. == Antibodies == BM-ca was produced by a recombinant CHO cell line and purified by a combination of several column chromatographies. Rituximab, (-)-Huperzine A ofatumumab, and infliximab, which are the ones used in clinical practice, were obtained from Hoffman-La Roche, GlaxoSmithKline, and Johnson & Johnson, respectively..