Publications

Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.

Sustained complement activation is an underlying pathologic driver in many inflammatory and autoimmune diseases. Currently approved anti-complement therapies are directed at the systemic blockade of complement. Consequently, these therapies provide widespread inhibition of complement pathway activity, beyond the site of ongoing activation and the intended pharmacodynamic (PD) effects. Given the essential role for complement in both innate and adaptive immunity, there is a need for therapies that inhibit complement in diseased tissue while limiting systemic blockade. One potential approach focuses on the development of novel fusion proteins that enable tissue-targeted delivery of complement negative regulatory proteins. These therapies are expected to provide increased potency and prolonged tissue PD, decreased dosing frequency, and the potential for improved safety profiles. We created a library of bifunctional fusion proteins that direct a fragment of the complement negative regulator, complement receptor type 1 (CR1) to sites of tissue injury. Tissue targeting is accomplished through the binding of the fusion protein to complement C3 fragments that contain a surface-exposed C3d domain and which are covalently deposited on tissues where complement is being activated. To that end, we generated a fusion protein that contains an anti-C3d monoclonal antibody recombinantly linked to the first 10 consensus repeats of CR1 (CR11-10) with the intention of delivering high local concentrations of this complement negative regulatory domain to tissue-bound complement C3 fragments iC3b, C3dg and C3d. Biochemical and in vitro characterization identified several fusion proteins that inhibit complement while maintaining the C3d domain binding properties of the parent monoclonal antibody. Preclinical in vivo studies further demonstrate that anti-C3d fusion proteins effectively distribute to injured tissue and reduce C3 fragment deposition for periods beyond 14 days. The in vitro and in vivo profiles support the further evaluation of C3d mAb-CR11-10 as a novel approach to restore proper complement activation in diseased tissue in the absence of continuous systemic complement blockade.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ T cells show that direct interaction between γδ T cells and immature B cells in the spleen support an “innate” transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ T cells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after in vivo depletion of γδ T cells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ T cells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ T cells an attractive target to treat autoimmunity.

Kawasaki disease (KD) is the leading cause of acquired heart disease in children. The cause remains unknown; however, epidemiologic and demographic data support a single preceding infectious agent may lead to KD. A variety of pathophysiologic responses have been proposed, including direct invasion of the coronary arteries, a superantigen response, and a post-infectious autoimmune phenomenon. A role for B cell responses during KD are supported by numerous findings including B cell specific markers identified in genome wide association studies. We have recently published data showing children with KD have similar plasmablast (PB) responses to children with infections. Since during other infections, cells expressing antibodies against the preceding infection are enriched in PBs, we sought to explore the specific antibodies encoded by PBs during KD. In one child we see a massive expansion in IGHV4–34 utilizing antibodies, which has been associated with autoimmunity in the past. We further explored this expansion of IGHV4–34 utilization during the peripheral PB rise with next generation sequencing (NGS) analysis and utilizing newer techniques of chromium chip single cell separation (10x Genomics®). We also utilized peptide array screening to attempt to identify an antigen to the most prolific clones.