Quantitative Spatial Profiling of Antibody Delivery and Target Engagement Using Optically Labeled Antibodies

Visualization of drug distribution in human tumors is critical for optimizing biologic therapeutics, yet current clinical workflows cannot colocalize drugs and biomarkers on the same formalin-fixed, paraffin-embedded section. Methods: We present a reproducible workflow that integrates near-infrared imaging of optically labeled therapeutic antibodies with multiplex immunostaining and computational registration to enable quantitative, single-cell analysis. Results: Using panitumumab–IRDye800CW, we demonstrated heterogeneous accumulation in epidermal growth factor receptor–positive regions and quantified penetration gradients relative to tumor architecture. Conclusion: We established a multiplex imaging platform that enables single-cell analysis of therapeutic antibody delivery and pharmacodynamics in human formalin-fixed, paraffin-embedded tissues.