Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action.

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.

Monoclonal antibodies 1B3 and 5C8 as probes for monitoring the integrity of the C-terminal end of soluble angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) is a membrane-anchored ectoprotein that is proteolytically cleaved, yielding an enzymatically active soluble ACE. Two mouse monoclonal antibodies, MAbs 1B3 and 5C8, were generated to the C-terminal part of human soluble ACE. MAb 1B3 recognized the catalytically active ACE, as revealed by ELISA and precipitation assays, whereas Western blotting and immunohistochemisty on paraffin- embedded sections using MAb 5C8 detected denatured ACE. MAb 1B3 showed extensive cross-reactivity, recognizing 15 species out of the 16 tested. The binding of this MAb to ACE was greatly affected by conformational changes induced by adsorption on plastic, formalin fixation, and underglycosylation. Furthermore, MAb 1B3 binding to the mutated ACE (Pro1199Leu substitution in the juxtamembrane region, leading to a fivefold increase in serum ACE level) was markedly decreased. MAb 5C8 detected all the known expression sites of full-size ACE using formalin-fixed and paraffin-embedded human tissues. The sequential epitope for MAb 5C8 is formed by the last 11 amino acid residues of soluble ACE (Pro1193-Arg1203), whereas the conformational epitope for 1B3 is formed by a motif within these 11 amino acid residues and, in addition, by at least one stretch that includes Ala837-His839 located distal to the sequential epitope. Our findings demonstrated that MAbs 1B3 and 5C8 are very useful for the study of ACE shedding, for identification of mutations in stalk regions, and for studying alternatively spliced variants of ACE. In addition, binding of MAb 1B3 is a sensitive determinant of the integrity of soluble ACE.

Selective rat lung endothelial targeting with a new set of monoclonal antibodies to angiotensin I-converting enzyme.

We demonstrated previously that monoclonal antibody (mAb) 9B9 to angiotensin-converting enzyme (ACE) accumulates selectively in the rat lung after systemic injection and thus is a powerful tool for immunotargeting therapeutic agents/genes to the lung microvasculature. Bearing in mind the tremendous research and therapeutic potential of lung immunotargeting via ACE, we generated a novel set of mAbs to rat ACE in order to enhance the repertoire of mAbs suitable for targeting drugs/genes to the rat lung. Five new mAbs recognizing different epitopes on rat ACE were examined for their efficacy to bind rat ACE both in vitro and in vivo. Gene delivery into cultured rat lung endothelial cells increased 30-50-fold after coating modified adenoviruses (containing Ig-binding domain) with mAbs to rat ACE. Radiolabeled mAbs specifically accumulated in the lung after systemic injection. mAb 1A2, 4H3 and 2E1 demonstrated the highest efficacy of lung uptake-around 50% of injected dose per gram of tissue; for mAb 1A2, the selectivity of lung uptake (ratio of lung to blood radioactivity) was 205. The effect of the mAbs on ACE shedding was epitope-specific: injection of mAb 1A2 and 4H3 did not change lung ACE activity, whereas injection of mAb 2E1 and 9B9 decreased rat lung ACE activity by 20%. None of the tested mAbs inhibited ACE activity in vitro. A new set of mAbs to rat ACE demonstrated highly efficient and selective lung accumulation and thus have the potential for targeting drugs/genes to the pulmonary vasculature in different rat models of lung diseases.