Epitope-dependent selective targeting of thrombomodulin monoclonal antibodies to either surface or intracellular compartment of endothelial cells.

Internalization of antibodies to thrombomodulin (TM) may provide a mechanism for intraendothelial targeting of drugs or genes. This study characterized three monoclonal antibodies against human TM (mAb 1009,1029, and 1045) and examined their internalization by human umbilical vein endothelial cells (HUVEC). It assessed binding of antibodies to recombinant human TM containing chondroitin sulfate (complete, cTM) and TM lacking chondroitin sulfate (incomplete, iTM). Direct RIA, indirect RIA, and ELISA and competitive ELISA show that (1) mAb 1009 binds to both cTM and iTM independently of divalent cations; (2) binding of mAb 1029 to iTM requires divalent cations, while binding to cTM is cation-independent; (3) mAb 1045 binds selectively to cTM independently of divalent cations. Binding of all three antibodies to the surface TM in HUVEC at 4 degrees C was similar by indirect immunostaining. In permeabilized HUVEC, however, mAb 1009 and 1029 provide brighter intracellular staining than mAb 1045. Uptake of (125)I-mAb 1009 by HUVEC at 37 degrees C was significantly higher than that of (125)I-mAb 1045. Low temperature markedly suppresses binding of (125)I-mAb 1009 to HUVEC, but has no effect on (125)I-mAb 1045 binding. About 80% of radiolabeled mAb 1045 bound to HUVEC at 37 degrees C could be eluted by acidic buffer from the cell surface, but only 40% of mAb 1009 and 1029 was elutable at these conditions. About 70-80 % of (125)I in cell lysates was TCA-soluble after HUVEC incubation with either mAb 1009 and 1029, but only 10 and 2.5% of (125)I was TCA-soluble in cell lysates and medium after 90 min incubation with (125)I-mAb 1045 at 37 degrees C. Therefore, HUVEC internalize and degrade an mAb that reacts with iTM, yet do not internalize an mAb that reacts selectively with cTM (mAb 1045). This result implies that either HUVEC do not internalize cTM constitutively or mAb 1045 suppresses TM internalization. Therefore, antibodies recognizing different TM epitopes might provide targeting of drugs to different cellular compartments.