BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

• Keywords
• Cancer cell migration, GAMG glioblastoma, HEK-Blue IL-6, IL-6, IL-6R blockers, Malignant melanoma, Migrastatics, Pancreatic carcinoma, Protein engineering,
• MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Cell Movement * drug effects   MeSH
• Cell Proliferation * drug effects   MeSH
• Receptors, Interleukin-6 * metabolism   MeSH
• Protein Binding drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IL6R protein, human MeSH Browser
• Receptors, Interleukin-6 * MeSH

IgA nephropathy (IgAN), the most prevalent primary glomerulonephritis worldwide, carries a considerable lifetime risk of kidney failure. Clinical manifestations of IgAN vary from asymptomatic with microscopic or intermittent macroscopic haematuria and stable kidney function to rapidly progressive glomerulonephritis. IgAN has been proposed to develop through a 'four-hit' process, commencing with overproduction and increased systemic presence of poorly O-glycosylated galactose-deficient IgA1 (Gd-IgA1), followed by recognition of Gd-IgA1 by antiglycan autoantibodies, aggregation of Gd-IgA1 and formation of polymeric IgA1 immune complexes and, lastly, deposition of these immune complexes in the glomerular mesangium, leading to kidney inflammation and scarring. IgAN can only be diagnosed by kidney biopsy. Extensive, optimized supportive care is the mainstay of therapy for patients with IgAN. For those at high risk of disease progression, the 2021 KDIGO Clinical Practice Guideline suggests considering a 6-month course of systemic corticosteroid therapy; however, the efficacy of systemic steroid treatment is under debate and serious adverse effects are common. Advances in understanding the pathophysiology of IgAN have led to clinical trials of novel targeted therapies with acceptable safety profiles, including SGLT2 inhibitors, endothelin receptor blockers, targeted-release budesonide, B cell proliferation and differentiation inhibitors, as well as blockade of complement components.

• MeSH
• Galactose MeSH
• Glomerulonephritis, IGA * diagnosis   MeSH
• Immunoglobulin A MeSH
• Antigen-Antibody Complex MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• galactosyl-deficient IgA1 MeSH Browser
• Galactose MeSH
• Immunoglobulin A MeSH
• Antigen-Antibody Complex MeSH

INTRODUCTION: Immunoglobulin A1 (IgA1) with galactose-deficient O-glycans (Gd-IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). Mucosal-tissue infections increase IL-6 production and, in patients with IgAN, are often associated with macroscopic hematuria. IgA1-secreting cell lines derived from the circulation of patients with IgAN, compared to those of healthy controls (HCs), produce more IgA1 that has O-glycans with terminal or sialylated N-acetylgalactosamine (GalNAc). GalNAc residues are added to IgA1 hinge region by some of the 20 GalNAc transferases, the O-glycosylation-initiating enzymes. Expression of GALNT2, encoding GalNAc-T2, the main enzyme initiating IgA1 O-glycosylation, is similar in cells derived from patients with IgAN and HCs. In this report, we extend our observations of GALNT14 overexpression in IgA1-producing cell lines from patients with IgAN. METHODS: GALNT14 expression was analyzed in peripheral blood mononuclear cells (PBMCs) from patients with IgAN and from HCs. Moreover, the effect of GALNT14 overexpression or knock-down on Gd-IgA1 production in Dakiki cells was assessed. RESULTS: GALNT14 was overexpressed in PBMCs from patients with IgAN. IL-6 increased GALNT14 expression in PBMCs from patients with IgAN and HCs. We used IgA1-producing cell line Dakiki, a previously reported model of Gd-IgA1-producing cells, and showed that overexpression of GalNAc-T14 enhanced galactose deficiency of IgA1, whereas siRNA-mediated GalNAc-T14 knock-down reduced it. GalNAc-T14 was localized in trans-Golgi network, as expected. CONCLUSIONS: Overexpression of GALNT14 due to inflammatory signals during mucosal infections may contribute to overproduction of Gd-IgA1 in patients with IgAN.

• Keywords
• GalNAc-T14, IL-6 cytokine, IgA nephropathy, inflammation,
• Publication type
• Journal Article MeSH

IgA nephropathy (IgAN) is an autoimmune disease in which poorly galactosylated IgA1 is the antigen recognized by naturally occurring anti-glycan antibodies, leading to formation of nephritogenic circulating immune complexes. Incidence of IgAN displays geographical and racial disparity: common in Europe, North America, Australia, and east Asia, uncommon in African Americans, many Asian and South American countries, Australian Aborigines, and rare in central Africa. In analyses of sera and cells from White IgAN patients, healthy controls, and African Americans, IgAN patients exhibited substantial enrichment for IgA-expressing B cells infected with Epstein-Barr virus (EBV), leading to enhanced production of poorly galactosylated IgA1. Disparities in incidence of IgAN may reflect a previously disregarded difference in the maturation of the IgA system as related to the timing of EBV infection. Compared with populations with higher incidences of IgAN, African Americans, African Blacks, and Australian Aborigines are more frequently infected with EBV during the first 1-2 years of life at the time of naturally occurring IgA deficiency when IgA cells are less numerous than in late childhood or adolescence. Therefore, in very young children EBV enters "non-IgA" cells. Ensuing immune responses prevent infection of IgA B cells during later exposure to EBV at older ages. Our data implicate EBV-infected cells as the source of poorly galactosylated IgA1 in circulating immune complexes and glomerular deposits in patients with IgAN. Thus, temporal differences in EBV primo-infection as related to naturally delayed maturation of the IgA system may contribute to geographic and racial variations in incidence of IgAN.

• Keywords
• Epstein-Barr virus, IgA nephropathy, IgA system maturation, age of infection, galactose-deficient IgA1, virus spread,
• MeSH
• Black or African American MeSH
• Black People MeSH
• Child MeSH
• Glomerulonephritis, IGA * epidemiology   ethnology   MeSH
• Immunoglobulin A MeSH
• Antigen-Antibody Complex MeSH
• Epstein-Barr Virus Infections * epidemiology   ethnology   MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Child, Preschool MeSH
• Herpesvirus 4, Human MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Geographicals
• Australia MeSH
• Names of Substances
• Immunoglobulin A MeSH
• Antigen-Antibody Complex MeSH

Immunoglobulin A nephropathy (IgAN), characterized by mesangial deposition of galactose-deficient-IgA1 (Gd-IgA1), is the most common biopsy-proven primary glomerulonephritis worldwide. Recently, an improved understanding of its underlying pathogenesis and the substantial risk of progression to kidney failure has emerged. The "four-hit hypothesis" of IgAN pathogenesis outlines a process that begins with elevated circulating levels of Gd-IgA1 that trigger autoantibody production. This results in the formation and deposition of immune complexes in the mesangium, leading to inflammation and kidney injury. Key mediators of the production of Gd-IgA1 and its corresponding autoantibodies are B-cell activating factor (BAFF), and A proliferation-inducing ligand (APRIL), each playing essential roles in the survival and maintenance of B cells and humoral immunity. Elevated serum levels of both BAFF and APRIL are observed in patients with IgAN and correlate with disease severity. This review explores the complex pathogenesis of IgAN, highlighting the pivotal roles of BAFF and APRIL in the interplay between mucosal hyper-responsiveness, B-cell activation, and the consequent overproduction of Gd-IgA1 and its autoantibodies that are key features in this disease. Finally, the potential therapeutic benefits of inhibiting BAFF and APRIL in IgAN, and a summary of recent clinical trial data, will be discussed.

• Keywords
• B-cell activating factor BAFF, IgA nephropathy, a proliferation-inducing ligand APRIL, atacicept, dual inhibition,
• Publication type
• Journal Article MeSH
• Review MeSH