Paired-agent imaging for detection of head and neck cancers

Abstract

Head and neck cancers overwhelmingly overexpress epidermal growth factor receptor (EGFR). This overexpression has been utilized for head and neck cancers using molecular targeted agents for therapy and cancer cell detection. Significant progress has been made in using EGFR-targeted fluorescent antibody and Affibody molecule agents for fluorescent guided surgery in head and neck cancers. Although success in achieving tumor-to-background ratio of 3-5 have been achieved, the field is limited by the non-specific fluorescence in normal tissues as well as EGFR specific fluorescence in the oral cavity. We propose that paired-agent imaging (PAI) could improve the contrast between tumor and normal tissue by removing the fluorescent signal arising from non-specific binding. Here, ABY-029 - an anti-EGFR Affibody molecule labeled with IRDye 800CW - and IRDye 680RD conjugated to Affibody Control Imaging Agent molecule (IR680-Aff_ctrl) are used as targeted and untargeted control agents, respectively, in a panel of head and neck squamous cell carcinomas (HNSCC) to test the ability of PAI to increase tumor detection. Initial results demonstrate that binding potential, a value proportional to receptor concentration, correlates well to EGFR expression but experimental limitations prevented pixel-by-pixel analysis that was desired. Although promising, a more rigorous and well-defined experimental protocol is required to align ex vivo EGFR immunohistochemistry with in vivo binding potential and fluorescence intensity. Additionally, a new set of paired-agents, ABY-029 and IRDye 700DX, are successfully tested in naïve mice and will be carried forward for clinical translation.

Keywords: ABY-029; Affibody Control Imaging Agent; Affibody molecule; IRDye 680RD; IRDye 700DX; IRDye 800CW; epidermal growth factor receptor; fluorescence guided surgery; head and neck cancer; paired-agent imaging.

Figure 1.

The paired-agent imaging (PAI) approach uses tissue transport of two fluorescent agents to quantify EGFR expression: 1) an untargeted Control Agent (ROI_C); and 2) an extracellular receptor Targeted Agent (ROI_T). K₁ and k₂ are the rates of extravascular leakage and tissue efflux, respectively; & k₃ and k₄, “on” and “off” rates of targeted tracer binding. Kinetic PAI: The Lammertsma equation where R₁ is equation to K₁/K₁’. Single Time Point PAI: describes the calculation of BP with single time point images at times >30 min post-injection. Binding Potential (BP): The key parameter, k₃/k₄, is proportional to B_avail, the concentration of available binding sites, and k_A, the receptor-target binding affinity (inversely proportional to k_D, the receptor-target dissociation constant).

Figure 2.

Experimental image collection for SCC-15 tumor demonstrating the experimental flow. Mice with subcutanteous SCC-15 tumors were injected with ABY-029 and IRDye 680RD-Affibody Control Imaging Agent. Fluorescent images were collected on the Odyssey CLx over an hour. The final images collected at 60 minutes are shown here for both ABY-029 and IRDye 680RD-Aff_ctrl. The corresponding and inherently aligned binding potential (BP) map is shown next to the aligned EGFR IHC performed in Matlab. The EGFR IHC image was color separated to isolate the brown stain only, and this brown stain was compared to the BP map, ABY-029 fluorescence and IRDye 680-Aff_ctrl fluorescence.

Figure 3.

EGFR expression measured in vitro correlates with in vivo BP measures. A) EGFR molecules per cell were quantified in vitro in five HNSCC cell lines using quantitative flow cytometry and are compared to the ‘gold standard’ of EGFR producing cell line, A431. B) High (SCC-15), moderate (SCC-25) and low (SCC-9) EGFR expressing cell lines were measured in vivo using PAI to determine the BP, which is proportional to EGFR concentration. C) The in vitro EGFR receptors per cell are plotted against the in vivo BP and a linear regression demonstrates that the two measures of EGFR correlate well.

Figure 4.

Correlation of EGFR stain intensity from immunohistochemistry with binding potential (left) and targeted ABY-029 (green, right) and non-targeted IR680-Aff_ctrl (red, right). BP displays a strong positive linear correlation with EGFR expression, while ABY-029 and IR680-Aff_ctrl display moderate and weak, negative linear correlations, respectively, with EGFR.

Figure 5.

Selection of clinically relevant agents for PAI. A) ABY-029 and IRDye 700DX are both in clinical trials and display very similar plasma excretion curves. B) Previous studies [27, 28] utilized EGF-IRDye 800CW and IRDye 700DX [27], and anti-EGFR Affibody molecule labeled with IRDye 800CW and IR680-Aff_ctrl [28]. The binding potential calculated in the same 4 tumor lines are linearly correlated with a strong, positive Pearson’s correlation (r = 0.98). C) The normalized fluorescence uptake in normal tissues (muscle, skin, fat) are similar. D) The binding potential map of normal tissue in a naïve mouse indicates that ABY-029 and IRDye 700DX be success paired-agents.