Date of Award

1-1-2016

Embargo Period

1-1-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Stephen Tomlinson

Second Advisor

Carl Atkinson

Third Advisor

Naren Banik

Fourth Advisor

Laura Kasman

Fifth Advisor

Abhay Varma

Abstract

The complement system is well known as a means of host defense but its excessive or prolonged activation can be counterproductive. Evidence points towards a dual role of complement in mediating spinal cord injury (SCI), however, the precise mechanism of its activation after SCI is still not known. In these studies, we attempted to elucidate the role of complement in SCI in a clinically relevant context by inhibiting the complement cascade at different activation/amplification steps. This knowledge is necessary to design optimal inhibitory strategies that can limit secondary injury while keeping the homeostatic effects of complement intact. Natural IgM antibodies function as innate immune sensors of injury via recognition of DAMPs (Damage associated molecular patterns). Therefore, we hypothesize that post-ischemic neoepitopes exposed as a result of SCI represent therapeutic targets both for blocking pathogenic IgM binding and for delivery of complement inhibitors. We demonstrated that self-reactive natural IgM antibodies, produced by B1 cells, bind to the injured spinal cord and activate complement. We identified 2 IgM mAbs (C2 mAb and B4 mAb) that were representative of this class of natural IgM Ab, and that recognized post-SCI DAMPs (or neoepitopes) and activated complement. Based on these data, we constructed a fusion protein consisting of a single chain Ab (scFv) derived from B4 mAb and linked it to the complement x inhibitor Crry, to form B4Crry. The complement inhibitor Crry inhibits all three complement activation pathways (classical, lectin and alternative) at the C3 activation step. We demonstrated that B4Crry specifically targeted to the injured spinal cord, inhibited complement activation, reduced tissue injury, and significantly improved functional recovery. Inhibiting only one complement pathway as opposed to all pathways would be optimal since it would be less immunosuppressive. We therefore investigated the role of the alternative pathway of complement activation in SCI using factor H (fH), an inhibitor specific for the alternative pathway. We also used an alternative targeting approach, and linked fH to complement receptor 2 (CR2). CR2 binds the complement activation product C3d, and thus targets to any site of complement activation. Thus, we characterized two therapeutic strategies that lead to improvement in recovery from paralysis after spinal cord injury. One of the strategies utilized is target specific and is aimed at targeting therapeutics to neoepitopes exposed on the injured spinal cord. This study identifies neoepitopes that represent novel therapeutic targets that could help prevent the need for systemic immune suppression. The second approach is pathway specific in which we target only the alternative pathway of complement activation. This study establishes the protective effects of inhibiting complement activation and amplification in the injured spinal cord.

Rights

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