AbstractThis study explores the impact of antibody surface charge on tissue distribution into various tissues including tumor. Tumor-bearing mice were dosed intravenously with a mixture comprising three antibodies engineered to carry negative charge patches, a balanced charge distribution, or positive patches, respectively (cassette dosing). Tissue levels were analyzed with a specific LC-MS/MS method. In addition, the antibody mix was administered to non-tumor bearing mice. Muscle and skin interstitial fluid were obtained by centrifugation and analyzed by LC-MS/MS. An in vitro endothelium model was explored for its feasibility to mimic the observed distribution differences.A balanced charge distribution was optimal in terms of total tumor exposure, while in other tissues, negatively charged and balanced charged antibodies gave similar results. In contrast, positive charge patches generally resulted in increased serum clearance but markedly enhanced tumor and organ uptake, leading to higher tissue-to-serum ratios. The uptake and availability in the interstitial space were confirmed by specific assessment of antibody levels in the interstitial fluid of the muscle and skin, with similar charge impact as in total tissue. The in vitro model was able to differentiate the transport propensity of this series of antibody variants. In summary, our results show the differential effects of charge patches on an antibody surface on biodistribution and tumor uptake. These insights may help in the design of molecules with biodistribution properties tailored to their purpose, and an optimized safety profile.