Fig. 2: Enhancing HIV-1 neutralization by NHR-targeting antibody through bispecific antibody-mediated prepositioning on host cells.

A Schematic of bispecific antibodies (bsAbs) generated by combining the antibody fragments of anti-NHR antibody D5_AR or anti-CD4 antibody iMab. mAb114 targeting unrelated Ebola glycoprotein is used as the control and is represented by the abbreviation ‘c’. A/B represents the antibody configuration, with antibody A contributing the knob arm (incorporating S354C and T366W substitutions) and antibody B contributing the hole arm (incorporating Y349C and T366S substitutions). B Flow cytometry to assess binding of bsAb to CD4-expressing TZM-bl cells and biotinylated NHR. Shown on the axes are the binding of the three antibodies to CD4 (probed with FITC-Fab fragment goat anti-human IgG, x-axis) and biotinylated NHR hydrophobic pocket mimetic IQN17 (probed with streptavidin-APC, y-axis). The configuration of the control Fab fragment is abbreviated as (c/) when on the knob arm and (/c) when on the hole arm. 10,000 cells were detected per measurement and results were plotted using FlowJo. The control shows binding to neither CCR5 nor NHR, while the bsAb binds both simultaneously. C Maximum % inhibition (left), IC₅₀ (middle), and IC₈₀ (right) for neutralization by iMab/D5_AR, its two control antibodies, and a mix of the two control antibodies, against a panel of 26 HIV-1 pseudotyped viruses representing diverse clades and tiers (see Table 1, source data are provided as a source data file). Each dot (n = 26) represents the average neutralization of biological duplicates of technical replicates for each pseudotyped virus. Maximum % inhibition values were calculated from the inhibition at the highest antibody concentration (50 μg/mL for iMab/D5_AR, and 50 μg/mL each for iMab/c and c/D5_AR in combination). IC₅₀ and IC₈₀ values were calculated using GraphPad Prism. Error bars indicate median +/- interquartile range. mAb114 Fab was used as a control Fab. The two-sided Wilcoxon-matched pairs signed test was used; **** indicates p < 0.0001. Black dotted lines indicate limit of quantification (50 μg/mL) of bsAb.