Journal: Nature Communications

Article Title: A monoclonal antibody that inhibits the shedding of CD16a and CD16b and promotes antibody-dependent cellular cytotoxicity against tumors

doi: 10.1038/s41467-025-64862-5

Figure Lengend Snippet: A Crystal structure of CD16a bound to Fc domain of hIgG1. Protein Data Bank 5YC5. B hIgG1-coated beads were incubated with 1 μg/mL biotinylated CD16a plus the indicated antibodies, followed by incubation with PE-labeled streptavidin and analysis by flow cytometry. See also Supplementary Fig. . C Jukart cells were engineered to express human CD16a and luciferase in response to the triggering of CD16a. The cells were treated overnight with antibodies, followed by analysis of luciferase activity in microplate plate reader. D Binding of antibodies to full length CD16a (indicated as “D1 + D2”), D1, or D2, as analyzed by ELISA. The indicated proteins were immobilized in multi-well plates, followed by incubation with the indicated antibodies. E The half maximal effective concentration (EC 50 ) values of the data in D, for F9H4. F , G F9H4 was digested with ficin to generate Fab and Fab2, which were validated for CD16a binding by ELISA with secondary antibody against murine Fab ( F ). CD16a shedding assay with the F9H4 fragments or controls (isotype and F9H4, which are undigested). MOLM13-CD16a WT cells were treated for 1 h with 10 μg/mL PMA and the indicated antibodies or fragments, followed by CD16a expression analyses through flow cytometry ( G ). H , I ELISA for the reactivity of the indicated mAbs to human CD16a protein ( H ). CD16a shedding assay with MOLM13-CD16a WT cells ( I ). The cells were treated for 1 h with 10 μg/mL PMA and the indicated mAbs, followed by CD16a expression analyses by flow cytometry. J F9H4 does not compete with 4H2F3 and 8E3B7 for CD16a binding. ELISA for the reactivity of the indicated antibodies to full length CD16a protein. The competition assay was done with the humanized version of F9H4, which compete against the murine version that serves as positive control. Data represent three ( B , D – J ) or two ( C ) independent experiments, are mean ± standard deviation ( C , I ) or standard error ( G ) of triplicates ( C , G , I ), and were analyzed by non-linear regression ( C , D , F – J ) and two-way ANOVA with Bonferroni’s test ( C , G , I ). *** p < 0.001. MFI = mean fluorescence intensity ( G , I ).

Article Snippet: The biotinylated secondary antibodies were as follows: anti-mIgG1 (RMG1-1, Biolegend catalog number 406604), anti-mouse IgG2a (RMG2a-62, Biolegend catalog number 407104), anti-mouse IgG2b (RMG2b-1, Biolegend catalog number 406704), mouse IgG3 (RMG3-1, Biolegend catalog number 406803), polyclonal anti-mouse IgG (catalog number BAF018, R&D Systems), polyclonal anti-mouse IgG Fab and Fab2 (315-005-006, Jackson ImmunoResearch Laboratories), and anti-human IgG Fc (M1310G05, Biolegend).

Techniques: Incubation, Labeling, Flow Cytometry, Luciferase, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Competitive Binding Assay, Positive Control, Standard Deviation, Fluorescence