Fig 2: Inhibition of IL-8 induced neutrophil chemotaxis.a Representative flow-cytometry results showing abN48-IgG1 and abN48-2-IgG1 (FITC-conjugated) specifically bind to human primary neutrophils. Blank: cells without hCXCR2 expression; iso-IgG1-FITC: irrelevant human IgG1 antibody conjugated with FITC. (3 independent biological replicates showed the same results) b Inhibition of abN48-2-IgG1 on IL-8 (10 nM) induced neutrophil chemotaxis was determined by a chemotaxis assay. Blank: media without IL-8 or inhibitor; IL-8: 10 nM IL-8; iso: 10 nM irrelevant human IgG1 antibody; MK: 1 µM small molecule CXCR2 inhibitor MK7123. The values of percentage (%) above the blue and green bars represent the percentage of inhibition of neutrophil chemotaxis induced by IL-8 at various concentrations of abN48-2-IgG1 and 1 µM MK7123, respectively. (n = 3 independent cell samples measured in a single experiment, data presented as dots overlapped with mean (column) ± s.d. (error bars). 3 independent experiments with different samples and antibody concentrations showed consistent results).

Fig 3: Species and subtype specificity of abN48-IgG1 and ab48-2-IgG1.a Representative images of co-localization of abN48-IgG1 or abN48-2-IgG1 to hCXCR2, hCXCR1, and mCXCR2 overexpressed on the membrane of U2OS cells. All test receptor proteins were fused with an mCherry fluorescent tag, and the immunocytofluorescent images were captured by confocal microscopy (3 images of every 3 samples were analyzed for each group and all gave consistent results). Scale bars (white) represent 50 µm. b Surface interactions of abN48-IgG1 and abN48-2-IgG1 with hCXCR2, hCXCR1, mCXCR2, rCXCR2, rbCXCR2, and mcCXCR2 overexpressed on U2OS cells. Flow-cytometry was used to measure the interactions between antibodies and the CXCR2 receptor proteins. Isotype-antibody (irrelevant human IgG1 antibody) was used as a negative control. For each sample, 3 repeats were carried out and all gave consistent results.

Fig 4: CXCR2 mediated cellular signaling pathways.a Activation of ß-arrestin signaling by IL-8, GRO-a, and the antibody ligands using a Tango reporter gene assay. b Inhibition of chemokine-induced ß-arrestin signaling by abN48-IgG1 and abN48-2-IgG1. The induction concentration of IL-8 (left) and GRO-a (right) were at their corresponding EC80 values. c Activation of Ca2+ influx by IL-8, GRO-a, and the abN48 antibody ligands using FLIPR measurement. d Inhibition of chemokine-induced Ca2+ influx by abN48-IgG1 and abN48-2-IgG1. The induction concentration of IL-8 (left) and GRO-a (right) were at their corresponding EC90 values. Data are represented as mean ± standard deviation. The calculated EC50 and IC50 values are listed next to the fitted curves. (For Tango and Ca2+ assay (a–d), n = 3 independent reporter cell samples measured in a single experiment, data presented as mean (center) ± s.d. (error bars). Each assay was repeated 3 times and all gave consistent results). e Antibody-dependent CXCR2 internalization. U2OS( + ) and U2OS(-) represent U2OS cells with and without surface hCXCR2 expression, respectively; CT represents the cytotoxin AL1-PE38KDEL; abN48 represents the antibody without cytotoxin; IT-iso, IT-1, and IT-2 represent the immunotoxins (ITs) assembled with an irrelevant human IgG1 antibody, abN48-IgG1 and abN48-2-IgG1 to the cytotoxin AL1-PE38KDEL, respectively. (n = 3 independent cell samples tested in a single experiment, data presented as mean (center) ± s.d. (error bars). This assay was repeated twice and gave consistent results.).