Figure 3.

Knob-in-hole-reversed eCD4-Ig variant diminishes cellular eCD4-Ig recombination with co-expressed anti-HIV VRCO1 IgG1 bNAb

(A) A non-reducing anti-IgG western blot from supernatants obtained from transiently transfected HEK293T cells with eCD4-Ig (WT), the anti-HIV bNAb VRCO1 IgG, or co-transfection of both plasmids. The red arrow identifies the VRCO1 and eCD4-Ig heterodimer band. A pictorial representation of the gel findings is shown: HC, IgG heavy chain; LC, IgG light chain; Fc domain showing CH2 and CH3 domain. Figures are not to relative scale. (B) A ribbon structural model showing the individual monomers (blue and tan) of the Fc portion of CH2 and CH3 domains of eCD4-Ig (see also Figure 1A), in which the CH3 dimerization interface was engineered to construct the eCD4-Ig knob-in-hole-reversed (KiHR). Shown are the KiHR mutations F405A and T394F in the CH3 dimerization interface (red and blue colored residues) which are predicted to sterically clash with CH3 domains of non-KiHR CH3 antibody domains. See materials and methods for additional information. (C) A non-reducing anti-IgG western blot analysis of HEK293T cell supernatants from cells transiently transfected with the plasmids eCD4-Ig or VRCO1, or co-transfected with the VRCO1 and eCD4-Ig-WT or VRCO1 and eCD4-Ig-KiHR plasmid combinations. The red arrow identifies the VRCO1 and eCD4-Ig-WT heterodimer band in the western blot lane. Note the presence of independent VRCO1 IgG and eCD4-Ig-KiHR bands in the co-expressed VRCO1 and eCD4-Ig-KiHR lanes and the lack of a heterodimer band. An illustration below the gel figure is a pictorial representation of the gel findings for eCD4-Ig-KiHR and VCR01 IgG lane.