Fig 2: 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.).

Fig 3: Alleviation of EAE symptoms in hCXCR2 knock-in mice by abN48-2-IgG1.a Schematic illustration of abN48-2-IgG1 treatment in an EAE mouse model. b Result of PK study of abN48-2-IgG1 in hCXCR2 knock-in mice (n = 4 mice per group). c, d EAE score and body weight of hCXCR2 knock-in mice during abN48-2-IgG1 treatment (n = 4 mice per group). e Comparison of neutrophils in peripheral blood of experimental mice. One blood sample was collected from each mouse in the indicated groups: “Naive” represents the group (n = 4 mice) without EAE stimulation; “isotype Fc” represents the group (n = 4 mice) treated with negative control (Fc); “PBS” represents the group (n = 4 mice) with EAE stimulation and PBS treatment; and “abN48-2-IgG1” represents the group (n = 4 mice) with EAE stimulation and abN48-2-IgG1 treatment (7.5 mg kg-1). “SB225002” represents the group (n = 4 mice) with EAE stimulation and daily treatment with CXCR2-selective inhibitor SB225002 (0.5 mg kg-1). For panel b to d, data are presented as mean (center) ± s.d. (error bars). For panel e, data are presented as dots overlapped with mean (column) ± s.d. (error bars). Two-tailed t-test was employed for the significance of differences. p < 0.0001 (p < 0.0001, ****) for t-test of “PBS” vs. “Naive” and “abN48-2-IgG1” vs. “PBS”, p = 0.0001 (p < 0.001, ***) for t-test of “abN48-2-IgG1” vs. “SB225002”. t-values and degree of freedom were referred to the “Methods” section.

Fig 4: 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.