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

Abstract

Vascular drug targeting may improve therapies, yet a thorough understanding of the factors that regulate effects of drugs directed to the endothelium is needed to translate this approach into the clinical domain. To define factors modulating the efficacy and effects of endothelial targeting, we used a model enzyme (glucose oxidase, GOX) coupled with monoclonal antibodies (anti-TM 34 or anti-TM 201 ) to distinct epitopes of thrombomodulin, a surface determinant enriched in the pulmonary endothelium. GOX delivery results in conversion of glucose and oxygen into H 2 O 2 leading to lung damage, a clear physiologic endpoint. Results of in vivo studies in mice showed that the efficiency of cargo delivery and its effect are influenced by a number of factors including: 1) The level of pulmonary uptake of the targeting antibody (anti-TM 201 was more efficient than anti-TM 34 ); 2) The amount of an active drug delivered to the target; 3) The amount of target antigen on the endothelium (animals with suppressed TM levels showed less targeting); and, 4) The substrate availability for the enzyme cargo in the target tissue (hyperoxia augmented GOX-induced injury). Therefore, both activity of the conjugates and biological factors control targeting and effects of enzymatic cargo. Understanding the nature of such "modulating biological factors" will hopefully allow optimization and ultimately applications of drug targeting for "individualized" pharmacotherapy.

Targeted Detoxification of Selected Reactive Oxygen Species in the Vascular Endothelium

“…This is important, because superoxide-mediated mechanisms, including NO consumption and production of highly injurious oxidants like peroxynitrite and hydroxyl radical in reactions with NO and H 2 O 2 , respectively, could contribute to tissue injury in the anti-TM/GOX-hyperoxia model. The lack of anti-PECAM/SOD protection, however, implies that these mechanisms are of relatively minor importance in this model and confirms that augmentation of anti-TM/GOX-induced injury by hyperoxia (Shuvaev et al, 2007a) is primarily due to enhancement of enzymatic activity of endothelium-bound GOX via increased supply of the ratelimiting substrate, oxygen. These results indicate that conversion of surplus superoxide into H 2 O 2 does not alleviate this type of oxidative injury, inflicted predominantly by H 2 O 2 , a more diffusible and long-living ROS that gives rise to highly injurious hydroxyl radical and hypochlorous acid.…”

“…However, in this "double-hit" model system, we believe it provides additional influx of oxygen substrate to GOX and thereby augments lung injury (Shu- 408 vaev et al, 2007a). Although we know that high levels of H 2 O 2 are produced and play an important role in inflicting maximal edematous lung injury within 4 h in this model (Shuvaev et al, 2007a), the role of superoxide had never been examined. This is important, because superoxide-mediated mechanisms, including NO consumption and production of highly injurious oxidants like peroxynitrite and hydroxyl radical in reactions with NO and H 2 O 2 , respectively, could contribute to tissue injury in the anti-TM/GOX-hyperoxia model.…”

“…In this model, pulmonary vascular oxidative stress was induced by intravenous injection of glucose oxidase conjugated with thrombomodulin antibody (anti-TM/GOX conjugate) in anesthetized C57BL/6 mice (The Jackson Laboratory). Anti-TM/GOX accumulates in the mouse lungs and generates H 2 O 2 from glucose, thereby causing an acute edematous lung injury at anti-TM/GOX dose of 1.0 g/g (Shuvaev et al, 2007a). To enhance injury by increasing the GOX substrate supply, animals were subjected to 80% O 2 in hyperoxic chamber for 4 h. Our previous study showed that this treatment aggravates oxidative stress and lung tissue injury in mice that received injections of anti-TM/GOX (Shuvaev et al, 2007a).…”

“…Anti-TM/GOX accumulates in the mouse lungs and generates H 2 O 2 from glucose, thereby causing an acute edematous lung injury at anti-TM/GOX dose of 1.0 g/g (Shuvaev et al, 2007a). To enhance injury by increasing the GOX substrate supply, animals were subjected to 80% O 2 in hyperoxic chamber for 4 h. Our previous study showed that this treatment aggravates oxidative stress and lung tissue injury in mice that received injections of anti-TM/GOX (Shuvaev et al, 2007a). The lungs were harvested and allocated for histopathology and immunostaining, and bronchoalveolar lavage (BAL) to test BAL fluid protein concentration.…”

Control of Endothelial Targeting and Intracellular Delivery of Therapeutic Enzymes by Modulating the Size and Shape of ICAM-1-targeted Carriers

“…This is the case for catalase that protects against vascular oxidative stress in cells and animal models. 18,23,24 Following internalization, anti-ICAM carriers traffic to lysosomes, where proteolysis terminates the therapeutic effect of catalase. 18,25 However, in an alternative therapeutic application, lysosomes represent an ideal destination of anti-ICAM carriers loaded with enzymes for replacement therapies for lysosomal storage disorders.…”