Figure 6.

ADCC of common 10-1074 Fc variants lacking fucose

10-1074 was produced in both HEK293T cells and FUT8 KO cell lines as wild-type IgG, LS mutant, LALA mutant, or a combination of LALA and LS mutants. Antibodies were purified by protein A column. (A) 3 μg IgG were loaded onto a 4%–12% bis-tris gel in duplicate. One was processed for Coomassie staining, the second was probed with AAL lectin to monitor the presence of α1-6 fucose. (B) SF162 gp140 trimer binding ELISA. Starting at an antibody concentration of 1 μg/mL followed by 3-fold serial dilutions, 10-1074 variants were incubated for ELISA measurement against SF162 gp140 trimer. High absorbance indicates high binding. (C) Neutralization curve of HIV-1 NL(AD8) with 10-1074 IgG1 starting at 0.625 μg/mL. The dashed line indicates 50% RLU representing 50% neutralization activity against HIV-1 NL(AD8). Lowest RLU indicates highest neutralization. (D) 10-1074 variants were tested for ADCC activity in triplicate. The dashed line indicates 50% RLU or 50% ADCC activity against HIV-1 NL(AD8)-infected target cells. ADCC was measured by the luciferase activity in HIV-infected cells after an 8-h incubation in the presence of a human CD16⁺ NK cell line and a serial dilution of antibodies. The loss of RLU indicates the loss of virus-infected cells during the 8-h incubation period and represents higher ADCC activity. Samples were analyzed for statistical significance compared to controls in (B)–(D) using a two-tailed paired t test.