Characterization of Novel P-Selectin Targeted Complement Inhibitors in Murine Models of Hindlimb Injury and Transplantation

Abstract

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

Screening and identification of biomarkers associated with the immune infiltration of intracerebral hemorrhage

“… 28 SELP (P‐selectin) as one of cell adhesion molecules discovered, mainly mediates platelet activation, endothelial cell adhesion and interaction with white blood cells, and is closely related to immune injury, inflammation, thrombosis, and tumor metastasis. 29 , 30 Studies on cerebrovascular diseases had found that when cerebrovascular endothelium was damaged, the expression of SELP in platelets and endothelial cells increased, and then binds with P‐selectin glycoprotein ligand‐1 (PSGL‐1) on leukocytes to activate signal transduction in leukocytes, causing leukocytes to release inflammatory factors and aggravate inflammatory responses. 31 Besides, PSGL‐1 and E/P‐selectins were essential for T‐cell rolling in inflamed CNS microvessels.…”

A Role for P-selectin and Complement in the Pathological Sequelae of Germinal Matrix Hemorrhage

“…We recently reported that following hindlimb ischemia and reperfusion in adult mice, 2.12Psel-Crry improved limb perfusion and increased bleeding time [22]. We thus considered that the difference in outcome between the 2.12 and 2.3 constructs in this hemorrhagic condition (2.12Psel-Crry worsens outcome and 2.3Psel-Crry improves outcome) may be related to an anti-coagulative effect of 2.12Psel-Crry.…”

“…2.3Psel-Crry and 2.12Psel-Crry are recombinant fusion proteins consisting of an anti-P-selectin single chain antibody (scFv) targeting domain linked to the complement inhibitor, Crry. Construction, expression, puri cation and in vitro characterization of these constructs was described previously [22]. The proteins were stored at -80C, and once thawed stored under sterile conditions at 4C for up to 2 weeks.…”

“…Tail bleed time in P13 C57BL/6J pups was measured 2 hours after their designated nal treatment (on P13) of PBS, 2.3Psel-Crry or 2.12Psel-Crry by a procedure previously described [22]. Brie y, pups were anesthetized with ketamine/xylazine mix and were placed in a prone position and a distal 5mm segment of the tail amputated.…”

“…Notably, a substantial uorescence signal remained in the brain at 72 hours post injection. Together with our previous data showing a fast-phase circulatory half-life of less than 1 hour for 2.3Psel-Crry [22], the data highlight an important feature of this site-targeted inhibitor, namely long-lived localized complement inhibition at the site of injury, with a minimal and rapidly declining effect on systemic complement activity.…”