A paired-agent fluorescent molecular imaging strategy is presented as a method to measure drug target engagement in whole tumor imaging. The protocol involves dynamic imaging of a pair of targeted and control imaging agents prior to and following antibody therapy. Simulations demonstrated that antibody “drug target engagement” can be estimated within a 15%-error over a wide range of tumor physiology (blood flow, vascular permeability, target density) and antibody characteristics (affinity, binding rates). Experimental results demonstrated the first in vivo detection of binding site barrier, highlighting the potential for this methodology to provide novel insights in drug distribution/binding imaging.
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