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

Abstract

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.

ICAM-1–targeted thrombomodulin mitigates tissue factor–driven inflammatory thrombosis in a human endothelialized microfluidic model

“…hTM/R6.5 bound to the surface of activated human ECs and restored APC generation capacity above that of uninflamed endothelium. hTM/R6.5 shares a number of desirable features with its mouse-specific analog, YN1/mTM, 19 and the use of an scFv fragment enables facile genetic fusion to therapeutic cargoes and mitigates the Fc and complement-mediated toxicities seen in clinical trials of the parental antibody, R6.5, or enlimomab. [44][45][46][47] To demonstrate the therapeutic efficacy of human-specific hTM/ R6.5, a microfluidic system consisting of entirely human components was developed to model TF-driven inflammatory thrombosis.…”

“…Second, the benefit of ICAM-targeted TM over its soluble counterpart supports the hypothesis that localization of the protein to the cellular membranes enhances its functional activity. 19,60,61 In fact, the difference in potency between these 2 agents may be more significant than their relative concentrations would suggest, as only a fraction of hTM/R6.5 molecules would have bound to the cell membrane. While ICAM-1 targeting of hTM/R6.5 was limited to hematopoietic and ECs in these studies, other cell types, such as epithelium, express ICAM-1, and may be both an important source of TF 24 and a valuable target of this approach.…”

“…(F) In vitro APC generation assay by cell-bound fusion protein, as described previously. 19 TNF-a-stimulated HUVECs show reduced APC generation capacity due to loss of surface TM, although some residual function is seen in comparison with cells treated with an anti-TM blocking mAb. For testing the functional activity of cell-bound hTM/R6.5, TNF-a-stimulated cells were incubated for 30 min at 37°C with various concentrations of fusion protein and then washed to remove nonspecifically bound protein prior to incubation with human thrombin (1 nM) and PC (100 nM).…”

“…In a murine model of acute lung injury, ICAM-1-targeted scFv/TM was found to facilitate interaction with endogenous EPCR, reduce inflammatory markers, and improve transendothelial plasma protein leakage. 19 While these studies demonstrated the potential therapeutic benefit of this approach, the effect on activation of coagulation was not examined.…”

“…[22][23][24] While ECs express TF under inflammatory stimuli in vitro, studies have failed to (E) hTM/R6.5 binds strongly to TNF-a-stimulated HUVEC but only minimally to nonstimulated ECs. Cell-based enzyme-linked immunosorbent assays were performed on live cells as previously described 19 using anti-FLAG-HRP to probe for cell-bound fusion protein. Graph shows mean 6 SD.…”