ENHANCED ENDOCYTOSIS IN CULTURED HUMAN BREAST-CARCINOMA CELLS AND IN-VIVO BIODISTRIBUTION IN RATS OF A HUMANIZED MONOCLONAL-ANTIBODY AFTER CATIONIZATION OF THE PROTEIN

For monoclonal antibody therapeutics to access target antigen in extra vascular compartments, an antibody drug delivery technology is require d that has the dual properties of 1) transendothelial migration of the antibody and 2) endocytosis of the antibody into the target cell. The se two objectives may be achieved with antibody cationization, and the present studies examine the feasibility of cationizing the humanized 4D5 monoclonal antibody directed against the p185(HER2) oncogenic prot ein. The cationized antibody binds to the p185(HER2) extracellular dom ain with an ED50 of 35 mu g/ml and inhibits SK-BRS cell proliferation similar to the native antibody. Confocal microscopy showed that althou gh there was binding of the native 4D5 antibody to the plasma membrane of SK-BR3 cells, this antibody was confined to the periplasma membran e space with minimal endocytosis into the cell. In contrast, robust in ternalization of the cationized 4D5 antibody by the SK-BR3 cells was d emonstrated by confocal microscopy. The systemic volume of distributio n of the cationized 4D5 antibody was Ii-fold greater than that of the native antibody. In summary, these studies show that a humanized monoc lonal antibody may be cationized with retention of antibody affinity f or the target antigen and biological activity, yet with a marked alter ation in the cellular distribution and pharmacokinetics in vivo.