Modulation of endothelial targeting by size of antibody-antioxidant enzyme conjugates

Abstract

Endothelial targeting of antioxidant enzymes attenuates acute vascular oxidative stress in animal studies. Superoxide dismutase (SOD) and catalase conjugated with antibodies to Platelet-Endothelial Cell Adhesion Molecule-1 (anti-PECAM/SOD and anti-PECAM/catalase) bind to endothelium, accumulate in the pulmonary vasculature, and detoxify reactive oxygen species. In order to define the role of conjugate size in the efficacy and specificity of endothelial targeting, we synthesized anti-PECAM/enzyme conjugates of controlled size (40nm-10,000nm). Binding of anti-PECAM/enzymes to endothelial cells increased with conjugate size from 300nm to 2μm (from 2.5 to 8.5% of bound fraction), and was specific, as conjugates did not bind to PECAM-negative cells. Pulmonary uptake of anti-PECAM/enzyme conjugates injected intravenously in mice also increased from 4.5 to 16% of injected dose for particles from 200 to 800nm. However, control conjugates larger than 300nm showed elevated non-specific pulmonary uptake, indicating that the targeting specificity of anti-PECAM/enzyme conjugates in vivo has a bell-shaped curve with a maximum close to 300-nm diameter. These results show that: i) the size of an antibody/enzyme conjugate modulates efficacy and specificity of targeting, and ii) a size optimum should be defined in vivo to account for parameters that are difficult to model in cell culture.

Figure 1

Modulation of streptavidin cross-linker input controls size of conjugates formed from biotinylated anti-PECAM and biotinylated enzymes. A. Anti-PECAM/SOD conjugates. B. Anti-PECAM/catalase conjugates. Inset, using highly biotinylated catalase forms larger particles. Size was measured using Dynamic Light Scattering (DLS). Representative curves are shown.

Figure 2

Preparation of conjugates using SATA/SMCC linker. A. Non-reducing SDS-gel electrophoresis of initial components IgG and catalase (Cat) and IgG/catalase conjugate preparation (conj). Coomassie staining of 4–15% gradient gel. B. Effects of reaction time on conjugate size. Catalase was modified at ratio catalase:SMCC 1:20 and IgG at IgG:SATA ratio 1:20. Conjugation reaction was performed for indicated time before size measurements. Inset: Effects of extent of catalase modification on conjugate size. Catalase was modified as indicated, while IgG:SATA molar ratio was fixed at 1:20. Proteins were mixed and conjugation reaction was performed for 60 min at room temperature before size measurements.

Figure 3

Stability of the anti-PECAM/enzyme conjugates and binding to endothelial cells. A, Conjugate size (●) and enzymatic activity (▲) after storage in 50% glycerol at −20ºC for at least 2 weeks. B. Binding of conjugates to endothelial cells. Cells grown to confluence were incubated with anti-PECAM/¹²⁵I-SOD conjugates of indicated size for 1 h at 37ºC, unbound material was washed with PBS and cells-bound conjugate was measured in a gamma-counter. Comparison of 300 nm (1, ●), 400 nm (2, ○), 700 nm (3, ▼) and 2.0 μm (4, △) conjugates binding to HUVEC vs PECAM-negative REN cells (5, ■). Mean±SD of four repeats are shown.

Figure 4

Blood and lung level of antibody-enzyme conjugates of various sizes in vivo. A–C. Blood level and pulmonary uptake of anti-PECAM/catalase conjugates in PECAM KO mice (PECAM KO, black bars) vs wild type mice (WT, gray bars). D–F. Blood level and pulmonary uptake of control IgG/SOD (black bars) vs anti-PECAM/SOD (gray bars) conjugates. ¹²⁵-I-labeled conjugates were injected IV, tissues were harvested in 1 h. A, 40 nm; B, 80 nm; C, 300 nm. D, 200 nm; E, 300 nm; F, 800 nm. * p<0.001 targeting vs. non-targeting conditions by t-test.

Figure 5

Lung uptake (A and C) and immunospecificity index (ISI; B and D) of anti-PECAM/catalase (A and B) and anti-PECAM/SOD (C and D) conjugates of various size. Radiolabeled conjugates were injected i.v. and tissue was harvested 1 h post-injection. The immunospecificity index was calculated as ratio of targeted vs non-immune counterparts. Tissue uptake is shown as mean±SEM (n=3–5). * p<0.001 targeted conjugate vs. non-immune counterparts by t-test.