Fig. 1: Construction and characterization of trispecific antibodies.

A Configuration of the trispecific antibody. Colored shades (blue or green) denote variable heavy and light chain domains, whereas grey shades denote constant heavy and light chain domains. VH/VL, variable fragments. B The indicated trispecific antibodies were analysed by SDS-PAGE once with gel image showing the bands at the appropriate molecular sizes for the two heavy chains above 50 kDa and two light chains above 25 kDa under reducing conditions (+) and a single predominant 175 kDa band under non-reducing conditions (−). C ELISAs showing that N6/αCD3-αCD28 binds to the CD4-binding site (CD4bs) of HIV Env, CD28 and CD3. The N6/αCD3-αCD28 and control trispecific antibodies at increasing five-fold concentrations were allowed to bind to either a resurfaced HIV Env fragment containing the CD4bs, CD28, human or cynomolgus CD3 that was coated on ELISA plates and the bound antibodies were detected using a HRP-conjugated anti-IgG probe. The data represents the mean values from two biologically independent experiments. D N6/αCD3-αCD28 binds to T cells and HIV Env on the cell surface. Human T cells, rhesus T cells and HIV-infected CEM cells were incubated with trispecific N6/αCD28-αCD3 and the control antibody, and bound antibodies were detected by a fluorescein isothiocyanate-conjugated anti-IgG probe. and (E) Neutralization IC50 titres (µg ml⁻¹) for the indicated trispecific N6/αCD3-αCD28 and control N6 protein against three representative HIV strains from clades (A, B and C). Source Data are provided as a Source Data file.