Fig. 2: Characterizing the new anti-FcγRI panel with focus on C01 and C04.
a Mid-point rooted phylogenetic trees for VL and VH chains of C01 (orange), C04 (red) and C03/C09/C10/C15/C16 (light orange), compared to sequences of clones 10.1 (blue), 611 and H22. b Titration curves of FITC-labeled anti-FcγRI antibodies of one representative experiment in duplo. c Binding of labeled anti-FcγRI mAbs or 10.1 on Ba/F3-FcγRI after pre-incubation with excess IVIg. d Anti-FcγRI antibody binding to PBMCs from healthy donors. hIgG1 (purple) was added as a control. Significance in c and d based on one-way ANOVA with Tukey’s test, **** p < 0.0001 compared to C03, C09, C10, C15, C16, and 10.1, N = 3, in duplicate. e Binding of C01 (circle, orange), C04 (square, red), and hIgG1 (diamond, purple) to fully human FcγRI and chimeric versions of FcγRI (i.e., mEC1 = only human EC1 swapped to mouse EC1). f Structure of the C01–FcγRI complex with domains and C01 CDR loops indicated. C01 binds exclusively to the EC2 domain of FcγRI. FcγRI is colored green, the C01 light chain purple, and the heavy chain orange. g Transparent surface of the C01 epitope on FcγRI in dark green and all epitope residues in stick representation and labeled. h Known binding regions of hIgG and 10.1 and proposed binding regions of own clones. Created in BioRender. Holtrop, T. (2025) https://BioRender.com/h1gt0ff. i, j Bind titration of C01 and C04 on FcγRIIa/b+ and FcγRIIIa/b+ cells. Anti-CD16 (triangle, light green) and anti-CD32 (upside-down triangle, blue) were taken along as a control. Data from one representative experiment in duplicate is shown. k Association and dissociation of C01, C04, and hIgG as measured by LigandTracer. A minimum of 4 runs per antibody were done, quantification of association and dissociation was done via TraceDrawer software (l). Boxplot showing distribution of obtained KD values. Midline indicates median, lower and upper margins of the boxes correspond with the 25th and 75th percentiles, and whiskers range from minimum to maximum values. C01/C04 is N = 6 and hIgG1 is N = 4. Significance determined with one-way ANOVA and Tukey’s test. m Area under the curve (AUC) of calcium flux on Ba/F3-FcγRI cells upon antibody binding and crosslinking with secondary antibody. M22 and H22 were used as positive controls. n Superoxide by IFN-γ-stimulated neutrophils in relative light units (RLU). Lines show the average of N = 3 measurements in triplicate for m22 and H22, and N = 6 for mAb 197, C01, C04, and 10.1. o AUC of superoxide, including baseline release (PBS), is indicated by a dotted line. Ordinary one-way ANOVA, with Tukey multiple comparison test. All line and bar data are presented as mean ± SEM. Source data are provided as a Source Data file.
