ENDOTHELIAL-CELLS INTERNALIZE MONOCLONAL-ANTIBODY TO ANGIOTENSIN-CONVERTING ENZYME

We investigated the fate of MAb 9B9, a monoclonal antibody to angioten sin-converting enzyme (ACE), which binds to endothelium both in vitro and in vivo. Using cultured human umbilical vein endothelial cells (HU VEC) and isolated pel fused rat lungs (IPL), we demonstrated specific and saturable binding of I-125-labeled MAb 9B9 at 4 degrees C [affinit y constant (K-d) = 20-50 nM, maximal number of binding sites (B-max) = 1.5-3.0 x 10(5) sites/cell]. When I-125-MAb 9B9 was bound to HUVEC at 37 degrees C, only 40% of cell-associated radioactivity was acid elut able, suggesting antibody internalization. This was confirmed by findi ng that 1) the amount of MAb 9B9 uptake at 37 degrees C was higher tha n at 4 degrees C both in HUVEC and IPL; 2) binding of I-125-labeled st reptavidin with HUVEC and IPL pretreated with biotinylated MAb 9B9 (b- MAb 9B9) was diminished in a temperature- and time-dependent fashion a t 37 degrees C; and 3)b-MAb 9B9 bound to HUVEC at 37 degrees C was fou nd intracellularly by ultrastructural analysis using streptavidin gold . Intracellular I-125-MAb 9B9 was found in microsomal fractions of lun g homogenate from IPL and after intravenous (iv) injections in rats. D egradation of internalized MAb 9B9 was minimal, since >90% of cell-ass ociated I-125 label remained precipitable by trichloracetic acid in HU VEC, IPL, and in vivo. Autoradiography of sodium dodecyl sulfate-polya crylamide gel electrophoresis of lung homogenates made as late as seve ral days after iv injections of I-l25-MAb 9B9 in rats demonstrated a p redominant band above 140 kDa. These data indicate that endothelial ce lls either in vitro or in vivo internalize the ACE ligand MAb 9B9 with out significant intracellular degradation. Therefore MAb 9B9 may be us eful for selective intracellular delivery of drugs to the pulmonary va scular endothelium after systemic administration.