Lung uptake of antibodies to endothelial antigens: key determinants of vascular immunotargeting

Abstract

Vascular immunotargeting is a mean for a site-selective delivery of drugs and genes to endothelium. In this study, we compared recognition of pulmonary and systemic vessels in rats by candidate carrier monoclonal antibodies (MAbs) to endothelial antigens platelet endothelial cell adhesion molecule (PECAM)-1 (CD31), intercellular adhesion molecule (ICAM)-1 (CD54), Thy-1.1 (CD90.1), angiotensin-converting enzyme (ACE; CD143), and OX-43. Tissue immunostaining showed that endothelial cells were Thy-1.1 positive in capillaries but negative in large vessels. In the lung, anti-ACE MAb provided a positive staining in 100% capillaries vs. 5-20% capillaries in other organs. Other MAbs did not discriminate between pulmonary and systemic vessels. We determined tissue uptake after infusion of 1 microg of (125)I-labeled MAbs in isolated perfused lungs (IPL) or intravenously in intact rats. Uptake in IPL attained 46% of the injected dose (ID) of anti-Thy-1.1 and 20-25% ID of anti-ACE, anti-ICAM-1, and anti-OX-43 (vs. 0.5% ID of control IgG). However, after systemic injection at this dose, only anti-ACE MAb 9B9 displayed selective pulmonary uptake (16 vs. 1% ID/g in other organs). Anti-OX-43 displayed low pulmonary (0.5% ID/g) but significant splenic and cardiac uptake (7 and 2% ID/g). Anti-Thy-1.1 and anti-ICAM-1 displayed moderate pulmonary (4 and 6% ID/g, respectively) and high splenic and hepatic uptake (e.g., 18% ID/g of anti-Thy-1.1 in spleen). The lung-to-blood ratio was 5, 10, and 15 for anti-Thy-1.1, anti-ACE, and anti-ICAM-1, respectively. PECAM antibodies displayed low pulmonary uptake in perfusion (2% ID) and in vivo (3-4% ID/g). However, conjugation with streptavidin (SA) markedly augmented pulmonary uptake of anti-PECAM in perfusion (10-54% ID, depending on an antibody clone) and in vivo (up to 15% ID/g). Therefore, ACE-, Thy-1.1-, ICAM-1-, and SA-conjugated PECAM MAbs are candidate carriers for pulmonary targeting. ACE MAb offers a high selectivity of pulmonary targeting in vivo, likely because of a high content of ACE-positive capillaries in the lungs.

Antibody-mediated lung endothelium targeting: in vivo model on primates

“…On average, only 10-20% of human and rat capillary endothelial cells in systemic circulation demonstrate ACE expression; whereas, virtually all endothelial cells in pulmonary capillaries strongly express ACE. 5,6 This finding explains why among several anti-endothelial mAbs studied (ICAM-1 (CD-54), PECAM (CD-31), Thy.1.1 (CD-90), ACE (CD-143)), anti-ACE mAbs demonstrated the most selective lung accumulation after systemic injection. 6 The selectivity of ACE expression in the lung appears to be fairly ubiquitous across mammals, as we have observed a similar pattern of ACE expression in pulmonary versus systemic circulation across all 26 species tested, including human and non-human primates (Franke et al, in preparation).…”

“…The observed high ACE level in the kidney appears to conflict with the absence of mAb 9B9 in kidney after in vivo antibody injection in all animals studied previously. [6][7][8][9][10] However, in the kidney, ACE is most highly expressed in the epithelium of proximal tubules and is found as well in brush border of intestine. 19 Thus, both of these areas are essentially inaccessible to monoclonal antibodies in circulation.…”

“…[6][7][8][9][10][11][12][13] It is likely that the large endothelial surface area, [20][21][22] and the consequent high ACE levels in the lung contributes to the selective accumulation of anti-ACE mAbs in the lung endothelium after systemic injection. [6][7][8][9][10][11][12][13] Another contributing factor to this accumulation is the heterogeneous distribution of ACE in human and rat endothelial cells along the vascular tree. On average, only 10-20% of human and rat capillary endothelial cells in systemic circulation demonstrate ACE expression; whereas, virtually all endothelial cells in pulmonary capillaries strongly express ACE.…”

“…Thus, despite similar affinities, mAbs to different epitopes of PECAM-1 demonstrated pronounced difference in internalization after binding with endothelial cell surface and subsequently, in pulmonary uptake. 6 From four studied monoclonal antibodies, recognizing different rat lung endothelial antigens on tissue slices, Western blotting or in cultured cells, only two of them demonstrated selective pulmonary uptake. 25 Therefore, only in vivo experiments can confirm the feasibility of different anti-ACE mAbs for ACE-dependent, antibody-directed lung endothelium targeting.…”

“…[3][4][5][6] The unique tissue distribution of endothelial ACE, along with preferential expression in lung capillaries, 5,6 also makes it an almost ideal target for therapy directed toward pulmonary endothelium.…”