Fig. 1: Binding of Bi-Ab32/16 and induced phenotypic alterations.

A Schematic illustrating the structure of the tetravalent bispecific antibody Bi-Ab32/16, comprising the IgG A32 antibody, targeting the gp120 target protein on HIV-infected CD4 + T cells, and the single-chain variable fragment (ScFv) CD16a directed towards the CD16 receptor expressed on NK cells. Created in BioRender. Genescà, M. (2025) https://BioRender.com/m03t574. B Representative flow cytometry plots depicting the binding of anti-HIVgp120 (A32 antibody, 5 µg/ml) and Bi-Ab32/16 (0.2 µg/ml) to CEM NKR CCR5+ cells coated with the HIV-1 Bal gp120 recombinant protein after 20 min incubation. Recognition of the respective antigens by the antibodies was determined using an anti-human secondary antibody targeting the heavy chains on human IgG antibodies, including A32. C Representative flow cytometry plots illustrating the binding of anti-CD16 (3G8 antibody) and Bi-Ab32/16 to primary isolated NK cells. The ability of the antibodies to recognize their cognate antigens was assessed using an anti-mouse secondary antibody recognizing the 3G8 antibody. D Summary graph of n = 7 independent experiments depicting the binding of anti-HIVgp120 and Bi-Ab32/16 to CEM NKR CCR5+ cells coated with the HIV-1 Bal gp120 recombinant protein after 20 min incubation. E Summary graph showing the binding of anti-CD16 (3G8 antibody) and Bi-Ab32/16 to primary isolated NK cells. N = 7 independent experiments are shown. F Representative gating strategy employed to identify cell doublets using FSC-H and FSC-A parameters (left), and subsequent analysis of the frequency of cell doublets expressing CD4 and CD16 (right) upon incubation with Bi-Ab32/16. G Optimized t-distributed stochastic neighbor embedding (opt-SNE) representations displaying the distribution of NK cell clusters based on the expression of different phenotypic markers in total (CD56 + ) NK cells by condition. Two conditions are depicted: control (R10 medium) and Bi-Ab32/16 following 24 h stimulation. H Heatmap depicting variations in the mean fluorescence expression of distinct NK cell receptors between control (R10 medium) and Bi-Ab32/16 conditions, corresponding to the different cell clusters identified in Fig. 1G and represented in different colors. I Violin plots showing the frequency of the distinct NK cell clusters identified in Fig. 1G among the distinct conditions. Statistical comparisons were performed using the Kruskal-Wallis test when required. Median with range (min-max) are shown. *p < 0.05; **p < 0.01.