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 activities 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.

Fig. 1

Drug delivery systems (DDS) for endothelial targeting of therapeutic enzymes. This schematic flowchart illustrates parameters that control targeting and effects of an enzyme cargo. Thus, DDS circulation is controlled by its size and charge. Targeting is controlled by features of a target determinant and DDS affinity. Sub-cellular localization of a drug is controlled by features of the selected target determinant, valence and size of DDS. These factors and biological features of the target tissue (Block V) modulate the outcome effects of targeted enzymes.

Fig. 2

Targeting of anti-TM/GOX formulations to pulmonary vasculature after intravenous injection in mice. Tissue levels of ¹²⁵Iodine 1 hour after IV injection of radiolabeled monoclonal antibodies and their formulations. Hatch bars: anti-TM₃₄, gray bars: anti-TM₂₀₁, black bars: irrelevant control IgG. Tissue distribution is shown as percent of injected dose per gram of tissue (%ID/g) of: ¹²⁵I-labeled anti-TM (A); anti-TM conjugated with ¹²⁵I-GOX via streptavidin-biotin cross-linking (B); and anti-TM-coated beads co-coated with ¹²⁵I-GOX (C). Unless specified otherwise, in this and subsequent figures the data are shown as mean ± SEM, n ≥ 3.

Fig. 3

Delivery efficacy and enzymatic activity of anti-TM/GOX formulations control their effect in the target tissue manifested in vivo by edematous lung injury. Panel A: protein level in the BAL fluid reflecting alveolar edema caused by oxidative stress by H₂O₂ produced by GOX were determined 4 hours after injection of indicated doses of anti-TM₃₄/GOX (black bars) or anti-TM₂₀₁/GOX (gray bars). Right bars show effects of 2.0 μg/kg control IgG/GOX (checkered bar) or anti-TM₂₀₁/bead/GOX (open bar). Asterisk: difference vs adjacent bar and background level of BAL protein in naïve mice (indicated by dash line) is significant at p<0.05. Panel B: pathological alterations in the lung tissue sections 4 hours after injection of 1.5 μg/kg of indicated preparations. Note accumulation of leukocytes and protein-rich edematous liquid in the alveoli depicted by asterisks. Panel C. Effect of biotinylation, conjugation and coating to polystyrene and PLGA nanoparticles on GOX activity. Inset: loading of radiolabeled GOX in polystyrene and PLGA nanoparticles: (1), 100 nm latex NC; (2), 200 nm latex NC; (3), 200 nm PLGA NC.

Fig. 4

Level of oxygen modulates effect of anti-TM/GOX in accumulated in mouse pulmonary vasculature. Panel A. Alveolar edema detected 4 hours after injection of anti-TM₃₄/GOX at room air (closed circles) vs 80% O₂ (open circles). Inset shows immunostaining for a marker of oxidative injury, nitrotyrosine, in lungs exposed to room air (left) vs hyperoxia (right). Panel B. Extent of lung injury 4 hours after injection of 1.25 μg/g of anti-TM₃₄/GOX is proportional to rate of oxygen supply. Asterisk: difference of hypoxia or hyperoxia vs room air is significant at p<0.05. Inset: hyperoxia augments level of a marker of lipid peroxidation, iPF2α isoprostane in the lung homogenates. Panel C. Kinetics of lung injury after the injection of 1.25 μg/g of anti-TM₃₄/GOX at room (closed circles) air vs 80% O₂ (open circles). Panel D. Alveolar edema 4 hours after injection of indicated doses of control non-targeted IgG/GOX (diamonds) vs anti-TM₃₄/GOX (open circles) or anti-TM₂₀₁/GOX (closed circles) conjugates at 80% O₂.

Fig. 5

Pathophysiological factors (oxidative stress induced by prolonged hyperoxia) modulate pulmonary TM expression and targeting of anti-TM. Panel A: Western-blotting of TM expression in the lung tissue homogenates in mice exposed to hyperoxia for 0, 24 or 48 hours. Panel B: Pulmonary uptake of ¹²⁵I-anti-TM 1 hour after injection in mice exposed for 48 hours to room air (black bars) or hyperoxia (light bars). Panel C: data shown in the panel B represented as organ-to-blood ratio, which accounts for differences in blood level of circulating radiolabeled antibody.

Fig. 6

Anti-TM/GOX pulmonary targeting and effect correlate with level of TM expression. Panel A: Western blotting analysis of lung TM and GOX four hours after anti-TM/GOX injection. Panels B and C: correlations of lung TM level vs GOX delivery (B) and GOX delivery vs lung injury (C).