Abstract 447: Development of CD47-nanomedicine as Novel Treatment for Specific Mitigation of Thrombospondin 1-Induced Vascular Dysfunction

Abstract

The trans-membrane receptor CD47 is considered a central relay of thrombospondin 1 (TSP-1) mediated responses in human cells/tissues, and ubiquitously serves as a marker of self on hematopoietic cells. Preliminary studies of TSP-1 binding with soluble human CD-47, serving as decoy recombinant protein (rhCD47p), demonstrated molar ratio ≥ 3 folds of rhCD47p abolished all TSP1-vascular CD47 receptor signaling. Accordingly, we attached multiple rh-CD47p ligands onto liposomes (NanoLip), as clinical prototype therapy. Novel rh-CD47p-NanoLip can avoid blood mononuclear cells, while binding many circulating TSP1 molecules, thus eliminating excessive plasma TSP-1, often implicated in pulmonary arterial hypertension (PAH) and ischemia-reperfusion injuries. Direct coupling of rh-CD47p, onto NanoLip surface was achieved using a sulfo-NHS-reaction between amino groups on rh-CD47p and corbodiimide-activated NanoLip, followed by column purification and physico-chemical analysis. Pharmaceutically, rh-CD47p-NanoLip formulation (av. size = 86±6.0nm) engaged 67±11μg/mL of protein, while retaining 86% of native rh-CD47p activity. Western blots and TSP-1-ELISA revealed dosimetric binding of tirmeric human TSP-1: rh-CD47p-NanoLip. Activated macrophages showed substantial decrease in phagocytosis of labeled rh-CD47p-NanoLip, compared to plain- and IgG-NanoLip controls (5- and 4- folds, respectively), indicating self-marker function. Conversely, binding of exogenous TSP-1 caused significant upturn of internalized rh-CD47p-NanoLip (p<0.05, n=4), due to TSP-1/nano-complex formation. Therapeutically, in isolated mouse thoracic aorta model, mimicking high plasma levels of TSP1 in PAH patients, titrated doses of CD47p-NanoLip neutralized TSP1-impaired vasodilation, back to the same level as controls (p<0.001, vs. TSP-1, n=4). This is the first report of its kind about CD47-based immunomodulatory nano-therapy specifically targeting TSP-1. The extracellular domains of native CD47 receptors of TSP1 were successfully “nano-modified” into a candidate systemic decoy pharmaceutical, rh-CD47p-NanoLip, to scavenge and eliminate pathologically elevated TSP-1 plasma levels, to abrogate TSP-1-associated vascular complications.