Description:
Technology: Inventors at MUSC have designed fusion proteins to treat inflammatory and other related pathologies by binding p-selectin (PSel) and modulating complement. Although targeting complement has shown great promise in treating numerous pathologies which has also led to the approval of multiple drugs (e.g. eculizumab, Berinert, Cinryze), targeting PSel offers a more targeted approach over complement because PSel is specifically expressed at sites of inflammation. For example, C3, the CR2 target, is expressed in healthy kidneys, spleen and lymph nodes, and on pathogens as a clearance mechanism that could be interfered with by CR2 inhibitors (e.g. eculizumab). To generate these fusion proteins, complement modulators were fused to a scFv for PSel that is non-blocking (NB.PSelscFv-Crry) or an scFv that blocks the interaction of P-selectin expressing endothelium or platelets with leukocytes that express a P-selectin ligand (B.PSelscFv-Crry), both of which have unique advantages described below.
For proof-of-concept studies, the inventors hypothesized fusion proteins would specifically traffic to the site of Ischemic Reperfusion Injury (IRI) due to the up-regulation of PSel following IRI. Administration of a PSel scFv linked to a C3 convertase inhibitor (Crry) reduced IRI (Figure 1), as measured by a reduction in injury, C3 deposition and neutrophil recruitment. The complement inhibitor utilized in the study is murine Crry, but any complement inhibitor of any species could be linked (eg. Human CR1, fH, C4BP, CD59, mAP44, or derivatives thereof).
Figure 1. Blocking and Non-blocking PSel scFv-Crry reduces IRI in vivo. A murine hindlimb IRI model was used to evaluate PSel scFv-Crry non-blocking (NB) and blocking (B) constructs. Sham, vehicle, and PSel scFv-Crry injections were performed via tail vein (n = 4). Histopathology revealed reduced skeletal muscle injury (A,D), complement deposition by C3d staining (B,E) and neutrophilic infiltrate (C,F) in mouse treated with either B or NB PSel scFv-Crry as compared to the vehicle control (PBS) and sham-injected mouse. Note the dose-dependent reduction from injury scores in both B and NB PSel scFv-Crry treated animals. No injury was observed in the sham mice
Additionally, the blocking construct displays dual activities by blocking both leyukocyte adhesion and inhibition of complement, with anti-coagulant activity, as demonstrated in a tail clip assay (Figure 2). Therefore, the non-blocking construct has use when anti-coagulant activity would not be advisable, while the blocking construct may have application in coagulopathies, including ischemic stroke patients eligible for thrombolysis therapy, post-transplant patients and thrombotic microangiopathy. Overall, these constructs have broad application for treating injury in general, IRI, inflammation, autoimmunity, alloimmunity, cancer, and any disease/condition in which the adhesion molecule P-selectin is expressed.
Figure 2. Tail clip assay demonstrating bleeding time of mice treated with B or NB fusion protein.
Overview: P-selectin is a cell surface glycoprotein expressed and upregulated by endothelial cells during inflammation and injury. P-selectin binds a mucin-like glycoprotein (PSGL) on the surface of myeloid cells including neutrophils and macrophages in addition to platelets. Binding then triggers the adhesion of inflammatory cells to the endothelium and subsequent activation of the cells and infiltration of local tissue. This phenomenon is implicated in several autoimmune and inflammatory diseases, including stroke, traumatic brain injury, ischemia and reperfusion injury, and transplantation. At the same time, complement activation is a major trigger of pathology in many diseases and disease states. In fact, complement activation on the surface of inflamed endothelium triggers a wide range of immune and inflammatory cascades that includes activation of immune cells, damage to tissue, release of cytokines, and expression of P-selectin on the endothelium. With a humanized version of the described constructs, complement inhibition would be an ideal potential treatment for patients presenting with any type of inflammatory condition, or disease/disease state where P-selectin is expressed at pathological site (would also include automimmune, ischemic, and transplantation-related pathology).
Publication: Manuscript under preparation
Applications: Reconstructive transplantation using vascularized composite allografts.
Advantages: Dual inhibition of p-selectin and C3 complement activation can decrease ischemia reperfusion injury and improve allograft transplantation
Key Words: Vascularized Composite Allotransplantation, Composite Tissue Allotransplantation, Allograft, Complement, P-selectin, Ischemia Reperfusion Injury
Inventors: Stephen Tomlinson, Michael Holers, Pablo Engel
Patent Status: US 16/572,260 filed 9.16.2019; US 17/335,355 filed 6.1.2021