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. 2020 Nov 17;9(4):63. doi: 10.3390/antib9040063

Figure 3.

Figure 3

FcγRIIIA binding and induction of ADCC by differentially engineered CD19 antibodies. (A) Binding of antibodies CD19-wt-CHO, CD19-EFTAE-CHO, CD19-wt-Lec13 and CD19-EFTAE-Lec13 to transfected BHK cells stably expressing human FcγRIIIA-158V (BHK-CD16-158V) or FcγRIIIA-158F (BHK-CD16-158F) alleles was analyzed by flow cytometry. Secondary FITC-coupled anti-human IgG Fc F(ab’)2 fragments were employed for detection. MFI, mean fluorescence intensity. (B) Induction of ADCC by antibody versions (concentration: 2 µg/mL) was investigated in 51Cr release experiments using Raji as target cells and human MNC as effector cells. Similarly designed variants of trastuzumab were used as controls. Bars represent mean values of specific lysis ± SEM. Significant differences between CD19 antibodies and HER2-specific control antibodies or the control reaction performed in the absence of any added antibody (no Ab) are indicated (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 n = 3). (C) Dose-dependent induction of ADCC by CD19 variants was analyzed using Raji (n = 3) or Ramos cells as targets and MNC as effector cells. Data points indicate mean values of specific lysis ± SEM. Statistically significant differences in ADCC between CD19 antibodies and the control antibody trastuzumab (IgG1) are indicated (*, p ≤ 0.05; **, p ≤ 0.01; n = 3). (D) Comparison of ADCC by the Fc double-engineered antibody CD19-EFTAE-Lec13 (purple) and by the CD20 antibody rituximab (black). Trastuzumab served as an additional negative control (IgG1). Raji cells were used as target cells and MNC served as effector cells. Mean values of specific lysis ± SEM are shown (n = 3).