A simplified fluorescence assay to estimate binding on- and off-rates for molecular targeted imaging agents

Accurate in vitro estimates of binding on- and off-rates (i.e., $k_{on}$ and $k_{\mathrm{off}}$ ) for any molecular targeted imaging agent can provide crucial information for predicting what agent dose and/or time(s)-of-imaging after agent administration will yield optimal contrast-to-noise. While there are several established in vitro methods for approximating $k_{on}$ and koff, they typically require multiple levels of dosing and equilibrium conditions to be met [1]. Here, a simplified method is described that enables $k_{on}$ and $k_{\mathrm{off}}$ to be estimated by direct fitting an analytical solution to a two-compartment binding model to cells stained with a single concentration of a fluorescent molecular imaging agent over a series of durations. Early simulations demonstrate that the method can yield errors of less than 10% in $k_{on}$ , $k_{\mathrm{off}}$ , and receptor quantification ( $B_{\text{max}}$ ) for staining solutions that are anywhere between 1-400% of the $B_{\text{max}}$ concentration, and results tend to improve when fitting is carried out on temporal data outside of the equilibrium condition. The method will be validated in various head and neck cancer human cell lines for the epidermal growth factor receptor- (EGFR-) targeted ABY-029-a fluorescently labelled affibody [2]-and for fluorescently labelled ultrasound nano-droplets seeded with multiple EGFR-targeted antibodies [3]. Both targeted agents are being used in translational studies for improving head and neck cancer tumor resection and lymph node biopsy, and the significant differences in size and overall chemistry highlight the breadth of utility provided by the described binding affinity assay.