Anti-EGFR Targeted Monoclonal Antibody Isotype Influences Antitumor Cellular Immunity in Head and Neck Cancer Patients

Abstract

Purpose: EGF receptor (EGFR) is highly overexpressed on several cancers and two targeted anti-EGFR antibodies which differ by isotype are FDA-approved for clinical use. Cetuximab (IgG1 isotype) inhibits downstream signaling of EGFR and activates antitumor, cellular immune mechanisms. As panitumumab (IgG2 isotype) may inhibit downstream EGFR signaling similar to cetuximab, it might also induce adaptive immunity.

Experimental design: We measured in vitro activation of cellular components of the innate and adaptive immune systems. We also studied the in vivo activation of components of the adaptive immune system in patient specimens from two recent clinical trials using cetuximab or panitumumab.

Results: Both monoclonal antibodies (mAb) primarily activate natural killer (NK) cells, although cetuximab is significantly more potent than panitumumab. Cetuximab-activated neutrophils mediate antibody-dependent cellular cytotoxicity (ADCC) against head and neck squamous cell carcinomas (HNSCC) tumor cells, and interestingly, this effect was FcγRIIa- and FcγRIIIa genotype-dependent. Panitumumab may activate monocytes through CD32 (FcγRIIa); however, monocytes activated by either mAb are not able to mediate ADCC. Cetuximab enhanced dendritic cell (DC) maturation to a greater extent than panitumumab, which was associated with improved tumor antigen cross-presentation by cetuximab compared with panitumumab. This correlated with increased EGFR-specific cytotoxic CD8⁺ T cells in patients treated with cetuximab compared with those treated with panitumumab.

Conclusions: Although panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at triggering antitumor, cellular immune mechanisms which may be crucial for effective therapy of HNSCC. Clin Cancer Res; 22(21); 5229-37. ©2016 AACR.

Figure 1. Cetuximab and panitumumab bind EGF receptor similarly but cetuximab activates PBMC to a greater extent than panitumumab

(A–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 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 PBMC from healthy donors were co-cultured with JHU-029 cells and treated with 10 μg/mL of cetuximab, panitumumab or isotype controls (IgG1 or IgG2) for 24h then surface expression of activation markers, CD69, CD16, CD107a and CD137 were measured by flow cytometry. PBMC treated with cetuximab express significantly higher activation markers compared to 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 PBMC or NK cell-dependent ADCC against HNSCC cells

(A) Whole PBMC co-cultured for 4h 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 to 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 co-cultured for 4h 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 pre-treated with either CD16 or CD32 blocking antibodies for 30 minutes, washed once, then labeled with ⁵¹Cr, then co-cultured with negatively isolated neutrophils for 4h in the presence of cetuximab or IgG1 control antibody (10 μg/mL) at an 80:1 E:T ratio. Cells pre-treated with CD16 blocking antibody show a reduction in cetuximab-activated neutrophil mediated ADCC compared with non-pretreated cells and cells pre-treated with CD32 blocking antibody. (C) Neutrophils from donors separated by FcγRIIIa and FcγRIIa genotype co-cultured for 4h 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⁺ positive selection) co-cultured with JHU029 and PCI15B HNSCC cells and treated with 10 μg/mL of cetuximab, panitumumab or isotype controls (IgG1 or IgG2) for 72h 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 co-cultured for 4h 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 co-cultured with NK cells and cetuximab or panitumumab for 48, measured by flow cytometry. HLA-DR expression on DC co-cultured with cetuximab is significantly higher than on DC co-cultured with panitumumab. (B) Mature DC, NK cells and JHU-029 (MAGE-3⁺) tumor cells were co-cultured 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 co-cultured in the presence of cetuximab was significantly higher than those co-cultured in the presence of panitumumab. EGFR-specific CTL frequencies of PBMC from patients on two 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 PBMC pre and post panitumumab. (D) The percentage of EGFR-specific CTL in patients treated with panitumumab did not significantly change post treatment. (E) Representative plots illustrating the frequency of EGFR-tetramer-positive CD8⁺ T cells in patient PBMC pre and post cetuximab. (E) Patients treated with cetuximab demonstrate significantly greater percentage of EGFR-specific CTL post treatment. Data are mean + SEM,*p<0.05, **p<0.01 ***p<0.001.