Vascular Immunotargeting to Endothelial Determinant ICAM-1 Enables Optimal Partnering of Recombinant scFv-Thrombomodulin Fusion with Endogenous Cofactor

<div><p>The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC) and thrombomodulin (TM), have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1) exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR), the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1) favors scFv/TM collaboration with EPCR. Indeed: i) endothelial targeting scFv/TM to ICAM-1 provides ∼15-fold greater activation of protein C than its PECAM-targeted counterpart; ii) blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii) anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.</p></div

APC generation by TM fusion proteins on non-endothelial REN cells with and without EPCR expression.

<p>(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.</p

Schematic representation of TM fusion proteins anchored to the endothelial plasmalemma.

<p>The proximity of ICAM-targeted TM to endogenous EPCR/Protein C may account for its enhanced activity <i>in vitro</i> and <i>in vivo</i>.</p

Anti-ICAM scFv/TM provides enhanced endothelial protection in a mouse model of lung inflammation/injury.

<p>(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.</p

Binding and activity of TM fusion proteins on mouse endothelial cells.

<p>(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.</p