Figure 4.

Fc Functions of AvFc

(A and B) SPR analysis of AvFc binding to FcγRI (A) and FcγRIIIa (B). Analysis was done in two independent experiments, and average K_D values are shown. (A) A representative FcγRI sensorgram. The raw (colored lines) and fitted (black lines) curves represent the concentrations of FcγRIA (1.2, 0.4, 0.13, 0.044, and 0.015 μg/mL, respectively, from top to bottom). K_D was determined based on the 1:1 binding kinetics. (B) A representative FcγRIIIa sensorgram. The raw data curves (colored lines) represent the concentrations of FcγRIIIA (100, 33.3, 11.1, 3.7, 1.2, and 0.41 μg/mL, respectively, from top to bottom). The K_D was determined based on steady state (inset). (C and D) Flow cytometry analysis of AvFc binding to FcγRI-expressing (C) and FcγRIIIa-expressing (D) TZM-bl cells. To eliminate background Avaren binding, samples were pre-incubated with yeast mannan. Representative flow histograms from triplicate analysis are shown. (E) HIV primary virus inhibition assay in PBMCs with aglycosylated (N200Q) AvFc. The assay was done in quadruplicate using four different PBMC preparations. (F) ADCVI activities of AvFc and VRC01. CD4⁺ lymphocytes were infected with HIV-1_92US657 at an MOI of approximately 0.05 for 72 h prior to the addition of AvFc (solid line) or VRC01 (broken line) and NK cells at an E:T ratio of 2:1. Data shown are representative of three independent triplicate experiments (expressed as mean ± SEM). (G) A schematic diagram showing AvFc’s anti-HIV mechanisms based on virion neutralization and Fc-mediated infected-cell killing.