Immunotargeting of glucose oxidase to endothelium in vivo causes oxidative vascular injury in the lungs

Abstract

Vascular immunotargeting is a novel approach for site-selective drug delivery to endothelium. To validate the strategy, we conjugated glucose oxidase (GOX) via streptavidin with antibodies to the endothelial cell surface antigen platelet endothelial cell adhesion molecule (PECAM). Previous work documented that 1) anti-PECAM-streptavidin carrier accumulates in the lungs after intravenous injection in animals and 2) anti-PECAM-GOX binds to, enters, and kills endothelium via intracellular H(2)O(2) generation in cell culture. In the present work, we studied the targeting and effect of anti-PECAM-GOX in animals. Anti-PECAM-GOX, but not IgG-GOX, accumulated in the isolated rat lungs, produced H(2)O(2,) and caused endothelial injury manifested by a fourfold elevation of angiotensin-converting enzyme activity in the perfusate. In intact mice, anti-PECAM-GOX accumulated in the lungs (27 +/- 9 vs. 2.4 +/- 0.3% injected dose/g for IgG-GOX) and caused severe lung injury and 95% lethality within hours after intravenous injection. Endothelial disruption and blebbing, elevated lung wet-to-dry ratio, and interstitial and alveolar edema indicated that anti-PECAM-GOX damaged pulmonary endothelium. The vascular injury in the lungs was associated with positive immunostaining for iPF(2alpha)-III isoprostane, a marker for oxidative stress. In contrast, IgG-GOX caused a minor lung injury and little (5%) lethality. Anti-PECAM conjugated with inert proteins induced no death or lung injury. None of the conjugates caused major injury to other internal organs. These results indicate that an immunotargeting strategy can deliver an active enzyme to selected target cells in intact animals. Anti-PECAM-GOX provides a novel model of oxidative injury to the pulmonary endothelium in vivo.

Factors modulating the delivery and effect of enzymatic cargo conjugated with antibodies targeted to the pulmonary endothelium

“…We tested distribution of the radiolabeled TM antibodies and GOX conjugates one hour after IV injection. Previous studies of immunotargeting of radiolabeled conjugates directed to highly accessible endothelial determinants such as TM, angiotensin-converting enzyme (ACE), PECAM-1, intercellular adhesion molecule 1 (ICAM-1) and GP90 showed that their maximum uptake in the lungs occurs within 15-30 min, followed by a short 15-30 min stable period and subsequent reduction of the pulmonary level observed at times more than one hour after IV injection [22,25,27,[46][47][48][49][50][51][52]. Therefore, the kinetics of lung edema development showing a maximum 4 hour after injection of a lower dose of the conjugate selected to avoid overt early lethality (Fig.4C) is not due to delayed accumulation of GOX in the lungs.…”

“…Protein iodination was performed using the iodination reagent IODO-beads (Pierce, Rockford, IL) in accordance with manufacturer's recommendations. Streptavidin cross-linker was used to produce b-anti-TM/SA/b-GOX or b-IgG/SA/b-GOX, as described in details in previous publications [19,22,24,25]. These conjugates are indicated as anti-TM 34 /GOX, anti-TM 201 /GOX or IgG/GOX in the text.…”

Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells

“…29,31,45 After SA and b-GOX were mixed and incubated for 1 hour on ice, the complex was then incubated with b-anti-TM, b-anti-PECAM, or b-IgG to form anti-TM/ GOX, anti-PECAM/GOX, or their nonimmune counterpart, IgG/GOX. When the molar ratios between SA/b-GOX and biotinylated antibodies were varied, sizes ranged from 100-to 2000-nm diameter, determined by DLS.…”

“…[23][24][25] Recent studies showed that monoclonal antibodies directed against these determinants (ie, anti-PECAM and anti-TM) could be used for immunotargeting to endothelial cells in vitro and in vivo. [26][27][28][29] Although endothelial cells poorly internalize anti-PECAM, anti-PECAM conjugated with streptavidin (SA) is readily internalized. 28 Both anti-PECAM/SA and anti-TM/SA serve as a carrier to deliver active enzymes and genes to pulmonary endothelial cells in intact animals.…”

“…28 Both anti-PECAM/SA and anti-TM/SA serve as a carrier to deliver active enzymes and genes to pulmonary endothelial cells in intact animals. [26][27][28][29][30][31][32] However, the carrier properties optimal for intracellular targeting to endothelium have not been established.Conceivably, carrier size is an important parameter for the intracellular uptake, yet this issue has not been systematically addressed in the literature. Available data show that optimal particle size threshold for intracellular uptake varies in different cell types.…”