The patho-physiological hallmark of acute lung injury (ALI) is severe derangement the interface of blood with the endothelial vascular lining. Endothelial cells (ECs) exert protective mechanisms including those mediated by thrombomodulin (TM), which inhibits thrombin's pathological activities in part through the production of activated protein C (APC) from its plasma precursor, via interactions between TM, thrombin, protein C, APC and the endothelial protein C receptor (EPCR) which is located in close proximity to TM in the endothelial plasmalemma. In ALI, TM and EPCR are functionally suppressed. Replenishment of this endothelial mechanism is an important therapeutic goal, but infusion of recombinant APC and TM did not improve the outcome of ALI, because problematic delivery and inability to partner with natural regulatory molecules limited effectiveness and lead to adverse effects. To solve these problems we fused TM to a single-chain variable fragment (scFv) of antibodies to PECAM, obtaining scFv/TM that provides protection superior to untargeted TM and APC in animal models of ALI. scFv/TM anchoring on endothelial cells in proximity to EPCR boosts protection. To circumvent the loss of endogenous EPCR typical of ALI, we use dual delivery of scFv/TM and scFv/EPCR, based on our recent serendipitous discovery of collaborative enhancement of endothelial binding of paired monoclonal antibodies directed to distinct PECAM epitopes. Here we will study the delivery, mechanisms and benefit/risk ratio of these fusions and embark on their clinical translation:
Aim 1 : Dual scFv/TM and scFv/EPCR targeting to endothelium. We will employ a combination of genetic and pharmacologic approaches to selectively modulate molecular regulation of the fusions in order to quantify their effects on ALI.
Aim 2 : Pre-clinical efficacy and safety in animals. Beneficial and adverse effects of anti-PECAM scFv/TM and scFv/EPCR will be characterized (vascular leakage, alveolar inflammatory response, morphologic and physiologic outcomes).
Aim 3 : Endothelial targeting scFv/TM in human lungs. To enable the clinical translation, we shall: A) Re-engineer the fusions using molecular elements applicable in humans; and, B) Study their targeting in isolated human lungs.
Our goal is to develop effective treatment for acute lung injury (ALI). The pulmonary vascular inflammation is a key target for therapeutic interventions in ALI. We have designed targeted biotherapeutics agents that recapitulate natural protective mechanisms of endothelial cells lining vascular lumen. In this grant we will define the molecular mechanisms of these prospective drugs, test them in animal models of ALI, and bring them to translation into the clinical domain.
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