Antibody Drug Conjugates (ADC’s) are one of the newest classes of biologics to be approved by the FDA for treating solid tumors. Development of these agents is exceedingly complex given the inter-related pharmacokinetics and pharmacodynamics of the biologic and small molecule. The antibody, linker, small molecule, and drug antibody ratio (DAR) must be optimized to achieve a therapeutic microdistribution within the tumor while maintaining safe systemic concentrations for ADC efficacy. Here we present a unique ratio imaging approach to track the distribution and metabolism of ADC’s from the whole animal to single-cell level. We developed a dual near infrared (NIR) fluorescent dye labeling technique to follow biologics at high temporal resolution from whole animal to organ, tissue, cellular, and subcellular spatial resolution to understand metabolism and distribution of ADC’s. Two NIR dyes with different physicochemical properties were selected so one dye washes out of cells after the protein is degraded (non-residualizing), while the other is retained (residualizing probe). For proof-of-principle, we quantified the metabolism of two antibodies, cetuximab and anti-A33, and one protein, EGF, in vitro and in vivo using a non-residualizing fluorophore to track intact protein and a residualizing fluorophore to measure cumulative uptake. As an application of this technique, we tracked the distribution of Kadcyla, an FDA-approved anti-HER2 antibody drug conjugate, in HER2 positive xenografts. The dual-tracers were used to image the whole mouse, quantify the biodistribution (%ID/g in each organ and tumor), measure tissue distribution with histology, and quantify the single-cell uptake in tumor cells. We demonstrated heterogeneous distribution in the tumor with widely varying single-cell distribution. We were also able to manipulate the distribution within the tumor using competing doses of Herceptin. This technique can be used to measure the impact of tissue and cellular heterogeneity on therapeutic efficacy with any antibody drug conjugate of interest.

Whole animal to single cell near infrared fluorescent imaging of antibody drug conjugate metabolism

251st National Meeting (2016)

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