Article Figures & Data
Figures
- Figure 1.
Cetuximab and panitumumab bind EGF receptor similarly, but cetuximab activates PBMCs to a greater extent than panitumumab. A and B, binding of cetuximab (IgG1)- and panitumumab (IgG2)-specific monoclonal antibodies (mAbs) to JHU-029 HNSCC cells. Cells were treated with increasing concentrations of either cetuximab or panitumumab (0.001–10 μg/mL) for 30 minutes at 4°C and then stained with either FITC-labeled Fc-specific Ab or EGFR Ab and analyzed by flow cytometry. Graphs show the percentage of FITC-positive cells obtained at each concentration of mAb. C, whole PBMCs from healthy donors were cocultured with JHU-029 cells and treated with 10 μg/mL of cetuximab, panitumumab, or isotype controls (IgG1 or IgG2) for 24 hours and then surface expression of activation markers, CD69, CD16, CD107a, and CD137, was measured by flow cytometry. PBMCs treated with cetuximab express significantly higher activation markers compared with cells treated with either control antibodies or panitumumab. Data are mean + SEM. **, P < 0.01; ****, P < 0.0001 cetuximab compared with panitumumab.
- Figure 2.
Greater triggering by cetuximab than by panitumumab of PBMCs or NK cell–dependent ADCC against HNSCC cells. A, whole PBMCs cocultured for 4 hours with 51Cr-labeled JHU-029 HNSCC cells coated with 10 μg/mL of cetuximab, panitumumab, or isotype controls (IgG1 or IgG2) at different E:T ratios (2.5:1, 5:1, and 10:1). Cetuximab significantly enhanced ADCC compared with panitumumab at an E:T ratio of 10:1. When this experiment was repeated using isolated NK cells (B), cetuximab significantly enhanced ADCC in comparison with panitumumab at all E:T ratios. Data are mean + SEM. *, P < 0.05; ****, P < 0.0001 cetuximab compared with panitumumab.
- Figure 3.
Cetuximab-activated neutrophil-mediated ADCC is enhanced in donors who are homozygous for FcγIIIa VV genotype and FcγIIa HH genotype. A, negatively isolated neutrophil cocultured for 4 hours with 51Cr-labeled JHU-029 HNSCC cells coated with 10 μg/mL of cetuximab, panitumumab, or isotype controls (IgG1 or IgG2) at different E:T ratios (10:1, 20:1, and 80:1). Cetuximab-activated neutrophils mediate ADCC above isotype controls, whereas panitumumab-activated neutrophils do not. B, JHU-029 HNSCC cells pretreated with either CD16 or CD32 blocking antibodies for 30 minutes, washed once, then labeled with 51Cr, and then cocultured with negatively isolated neutrophils for 4 hours in the presence of cetuximab or IgG1 control antibody (10 μg/mL) at an 80:1 E:T ratio. Cells pretreated with CD16 blocking antibody show a reduction in cetuximab-activated neutrophil-mediated ADCC compared with non-pretreated cells and cells pretreated with CD32 blocking antibody. C, neutrophils from donors separated by FcγRIIIa and FcγRIIa genotype cocultured for 4 hours with 51Cr-labeled JHU-029 HNSCC cells coated with 10 μg/mL of cetuximab, panitumumab, or isotype controls (IgG1 or IgG2) at 80:1 E:T ratio. Neutrophils from FcγRIIIa VV donors demonstrate significantly enhanced ADCC activity compared with VF and FF donors. D, neutrophils from FcγRIIa HH donors mediate enhanced ADCC compared with HR and RR donors. Data are mean + SEM. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.
- Figure 4.
Panitumumab activates CD32 receptors on monocytes to a greater degree than cetuximab but does not induce ADCC against HNSCC cells. Surface activation markers, CD32 (A) and CD80, CD86 (B) of isolated monocytes (CD14+ selection) cocultured with JHU029 and PCI15B HNSCC cells and treated with 10 μg/mL of cetuximab, panitumumab, or isotype controls (IgG1 or IgG2) for 72 hours were measured by flow cytometry. Monocytes treated with panitumumab activate surface CD32 to a greater extent than those treated with cetuximab as demonstrated by downregulation of surface CD32. There is no significant difference in CD80 or CD86 expression on monocytes treated with cetuximab or panitumumab. C, CD14+ cells cocultured for 4 hours with 51Cr-labeled JHU-029 HNSCC cells coated with 10 μg/mL of cetuximab, panitumumab, or isotype controls (IgG1 or IgG2) at different E:T ratios (5:1, 10:1, and 20:1). Results demonstrate that monocytes did not mediate ADCC above isotype controls in the presence of either cetuximab or panitumumab. Data are mean + SEM. *, P < 0.05 cetuximab compared with panitumumab.
- Figure 5.
Cetuximab enhances adaptive cellular immune responses to a greater extent than panitumumab. A, HLA-DR expression on CD11c+ DC cocultured with NK cells and cetuximab or panitumumab for 48 hours, measured by flow cytometry. HLA-DR expression on DC cocultured with cetuximab is significantly higher than on DC cocultured with panitumumab. B, mDC, NK cells, and JHU-029 (MAGE-3+) tumor cells were cocultured in the presence of no antibody, cetuximab (10 μg/mL), or panitumumab (10 μg/mL) at a 1:1:1 ratio for 48 hours. DC were then stained using 12B6 antibody and expression of MAGE-3 was analyzed by flow cytometry. MAGE-3 expression on DCs cocultured in the presence of cetuximab was significantly higher than those cocultured in the presence of panitumumab. EGFR-specific CTL frequencies of PBMCs from patients on 2 clinical trials employing chemoradiotherapy combined with either cetuximab or panitumumab measured by flow cytometry. C, representative plots illustrating the frequency of EGFR tetramer–positive CD8+ T cells in patient PBMCs pre- and post-panitumumab. D, percentage of EGFR-specific CTL in patients treated with panitumumab did not significantly change posttreatment. E, representative plots illustrating the frequency of EGFR tetramer–positive CD8+ T cells in patient PBMCs pre- and post-cetuximab. F, patients treated with cetuximab demonstrate significantly greater percentage of EGFR-specific CTL posttreatment. Data are mean + SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Additional Files
Supplementary Data
- Supplementary figure legends - Legends for Supplementary Figures 1, 2 and 3
- Supplementary Figures 1, 2, 3 - Sup Fig 1. Patient demographic data for clinical trials UPCI 08-013 and UPCI 06-120. Sup Fig 2.Flow cytometric plots from representative donor PBMC showing percentages of CD14+ and CD56+ Sup Fig 3. Flow cytometric histogram illustrating the relative EGFR expression
Volume 22, Issue 21
