ADCC activity was calculated according to the following formula: [ADCC activity (%)] = ([Fluorescence with antibody] ? [Fluorescence without antibody])/([Maximum Fluorescence] ? [Fluorescence without antibody]) 100, where Maximum Fluorescence is the value after treatment of target cells with 1% Triton X-100

ADCC activity was calculated according to the following formula: [ADCC activity (%)] = ([Fluorescence with antibody] ? [Fluorescence without antibody])/([Maximum Fluorescence] ? [Fluorescence without antibody]) 100, where Maximum Fluorescence is the value after treatment of target cells with 1% Triton X-100. ofatumumab (C and F) in SU-DHL-4 (ACC) and RC-K8 (DCF) cells. Each point represents the imply SD (= 4). Physique S3. Combination effects of anti-CD20 antibodies with hydroxydaunorubicin in the anti-cell proliferation assay. Effects of hydroxydaunorubicin at numerous concentrations were examined in the presence of 0 (?), 0.1 (), 1 (?), and 10 g/mL (?) of BM-ca (A and D), rituximab (B and E), or ofatumumab (C and F) in SU-DHL-4 (ACC) and RC-K8 (DCF) cells. Each point represents the imply SD (= 4). Physique S4. Combination effects of anti-CD20 antibodies with cisplatin in the anti-cell proliferation assay. Effects of cisplatin at numerous concentrations were examined in the presence of 0 (?), 0.1 (), 1 (?), and 10 g/mL (?) of BM-ca (A and D), rituximab (B and E), or ofatumumab (C and F) in SU-DHL-4 (ACC) and RC-K8 (DCF) cells. Each point represents the imply SD (= 4). Physique S5. Common representations of gating of lymphocytes (A), histogram without antibody (B), that with BM-ca-FITC (C), and that with rituximab-FITC (D), in circulation cytometry analysis of peripheral blood of monkeys (rhesus monkey; animal no. 1). M1 and M2 are negative and positive populations, respectively. Physique S6. Sequences of cDNAs encoding two different types of CD20 molecules in cynomolgus monkeys. In the a.a. 160 = Leu molecule, the nucleotide position 479 is usually T; whereas in the a.a. 160 = Pro molecule, it is C. Physique S7. Reactivity of BM-ca, rituximab, ofatumumab, and infliximab with CD20 molecule expressed on the surface of CHO cells in the ELISA assay under the same assay conditions as in Physique 8. Table S1. Effect of combination of anti-CD20 antibodies and malignancy chemotherapeutics in SU-DHL-4 cells. Table S2. Effect of combination of anti-CD20 antibodies and malignancy chemotherapeutics MLN9708 in RC-K8 cells. cam40002-0130-sd1.pdf (2.2M) GUID:?3E1991A2-8B86-4F2E-AF50-98634BB26BD2 Abstract Cellular activity of BM-ca, a novel humanized anti-CD20 antibody, was quantitatively compared with that of two other anti-CD20 antibodies utilized for clinical practice, rituximab and ofatumumab. The results of a complement-dependent cytotoxicity (CDC) assay revealed that this 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, even though effective concentrations against SU-DHL-4 cells were almost the same among these three antibodies, the MLN9708 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 malignancy 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 this responders and nonresponders experienced Leu/Pro (hetero) and Leu/Leu (homo) MLN9708 at amino acid MLN9708 (a.a.) position 160, respectively, suggesting that this 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 156C166, not shared with rituximab and ofatumumab. These results suggest BM-ca to be a encouraging anti-CD20 antibody having superior properties and realizing a unique epitope. = 2). ADCC activities of anti-CD20 antibodies Dose-dependent comparison of ADCC activities among the three anti-CD20 antibodies gave the results shown in Physique 2. Although there existed no significant differences in ADCC activities in RC-K8 cells, in SU-DHL-4 cells BM-ca lysed a larger quantity of target cells (about 25%) than the two other antibodies (about 10%) above plateau concentrations. The effective concentration range was almost the same among these three antibodies. Open in a separate window Physique 2 ADCC activity of BM-ca (?), rituximab (), ofatumumab (?), and infliximab (?) in RC-K8 (A) Mouse monoclonal to EhpB1 and SU-DHL-4 (B) cells. RC-K8 (40,000) and SU-DHL-4 (20,000) cells prelabeled with Calcein were.