Figure 1.
Localization of PECAM-1, ICAM-1, and EPCR on MS1 mouse endothelial cells.
(a) Immunofluorescence images demonstrate superior co-localization of ICAM-1 and EPCR, as compared to PECAM-1 and EPCR. (b) Radioimmunoassay data using 125I-labeled anti-PECAM and anti-ICAM parental antibodies to determine affinity and number of binding sites per endothelial cell.
Figure 2.
Binding of ICAM and PECAM-targeted scFv and scFv/TM fusion proteins.
Cell based ELISAs show binding of (a) anti-PECAM scFv and anti-PECAM scFv/TM fusion protein to PECAM expressing REN cells, and (b) anti-ICAM scFv and anti-ICAM scFv/TM fusion protein to ICAM expressing REN cells. No significant binding is seen to wild type REN cells. Experiments were done in triplicate (each point shown represents three wells), with SD shown. EC50 is shown for each curve.
Figure 3.
APC generation by TM fusion proteins on non-endothelial REN cells with and without EPCR expression.
(a) anti-PECAM scFv/TM and anti-ICAM scFv/TM activate protein C while bound to PECAM and ICAM-expressing cells, respectively. Minimal APC is generated on wild type REN cells, presumably due to lack of binding. (b) A ∼4-fold increase in APC generation is seen when PECAM and ICAM-targeted TM fusion proteins are anchored to cells which stably express mouse EPCR (i.e. REN-PECAM-EPCR and REN-ICAM-EPCR cells), as compared to EPCR-negative counterparts. All experiments were done in triplicate. Data shown are mean ± SD.
Figure 4.
Binding and activity of TM fusion proteins on mouse endothelial cells.
(a) Anti-PECAM scFv/TM and (b) anti-ICAM scFv/TM bind to their respective ligands on MS1 cells. Binding is inhibited by excess of parental anti-PECAM-1 and anti-ICAM-1 antibodies. (c) Anti-ICAM scFv/TM demonstrates ∼15-fold greater activity per binding site on MS1 cells, as compared to its PECAM-anchored counterpart. (d) Antibody blockade of EPCR results in a ∼50% decrease in APC generation by endogenous TM and anti-ICAM scFv/TM, but not anti-PECAM scFv/TM. All experiments were done in triplicate. Data shown are mean ± SD.
Figure 5.
Anti-ICAM scFv/TM provides enhanced endothelial protection in a mouse model of lung inflammation/injury.
(a) Timeline of intratracheal LPS lung injury model. In experiments assessing endothelial barrier dysfunction, a tracer amount of 125I-labeled albumin was injected 5 minutes prior to LPS administration. (b) Concentration of the chemokine MIP-2 in bronchoalveolar lavage (BAL) fluid. (c) mRNA transcript levels of pro-inflammatory cell adhesion molecules, VCAM-1 and E-selectin, in lung homogenate. (d) Endothelial barrier dysfunction, as measured by leakage of 125I-labeled albumin from blood into lung interstitium and/or alveolar space. All data shown are mean ± SD, with number of animals as shown.
Figure 6.
Schematic representation of TM fusion proteins anchored to the endothelial plasmalemma.
The proximity of ICAM-targeted TM to endogenous EPCR/Protein C may account for its enhanced activity in vitro and in vivo.