Development of specific peptide reagents for serologic monitoring of Exostosin autoantibodies in membranous lupus nephritis

膜性狼疮肾炎外骨蛋白自身抗体血清学监测特异性肽试剂的开发

• 批准号: 10545924
• 负责人: Christopher P Larsen
• 金额: $ 25.94万
• 依托单位: NEPHROPATHOLOGY ASSOCIATES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-05 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10545924
• 关键词: Address; Affinity; Antibodies; Antigen-Antibody Complex; Antigens; Autoantibodies; Autoantigens; Autoimmune; Bacteriophage M13; Bacteriophages; Binding; Biocompatible Materials; Bioinformatics; Biological Assay; Biopsy; Biopsy Specimen; Cells; Clinic; Clinical; Deposition; Development; Diagnosis; Diagnostic; Diagnostic Reagent; Diagnostic tests; Dialysis procedure; EXT1 gene; Enzyme-Linked Immunosorbent Assay; Etiology; Exhibits; Freezing; Future; G-substrate; Healthcare Systems; Immobilization; Immunoglobulin G; Incidence; Incubated; Individual; Injury; Kidney Failure; Laboratories; Libraries; Lupus Nephritis; Mass Spectrum Analysis; Membrane; Membranous Glomerulonephritis; Methods; Molecular; Monitor; Movement; Nephrology; Nephrotic Syndrome; Patient Care; Patients; Pattern; Peptides; Phage Display; Phase; Phospholipase A2; Population; Proteins; Proteomics; Provider; Publishing; Reagent; Recombinant Proteins; Recombinants; Reporting; Research; Risk; Sampling; Sensitivity and Specificity; Sequence Analysis; Serology; Serology test; Serum; Signal Transduction; Specificity; Stains; Subgroup; Surveys; Systemic Lupus Erythematosus; Testing; Thrombospondins; Tissues; Transplantation; Transplantation Surgery; base; biobank; bioinformatics pipeline; experience; kidney biopsy; laser capture microdissection; next generation sequencing; novel; phase 2 testing; precision medicine; programs; protein aminoacid sequence; receptor; screening; success; synthetic peptide; treatment response

Project Summary Up to half of patients with systemic lupus erythematosus (SLE) will develop lupus nephritis (LN), and nearly a third of LN patients will develop kidney failure requiring dialysis or transplantation. One type of LN, membranous lupus nephritis (MLN) exhibits similar clinical hallmarks and patterns of injury as compared to non- lupus associated membranous nephropathy (MN). MN and MLN are largely diagnosed through histopathological analysis of kidney biopsies. Moreover, autoantigens have been identified in MN, with M-type phospholipase A2 receptor (PLA2R) autoantibodies present in nearly 70% of cases and thrombospondin type-1 domain containing 7A (THSD7A) autoantibodies present in approximately 2% of cases. Given the strong connection between these autoantibodies and MN, there is significant movement toward eliminating kidney biopsies in patients showing serological positivity for autoantibodies against PLA2R and THSD7A. Moreover, serologic tests specific for the inciting autoantibody (anti-PLA2R or anti-THSD7A) have proven to be highly valued by nephrologists for monitoring response to therapy and guiding patient care. Reducing the requirement for renal biopsy and progressing towards a precision medicine approach represent dramatic benefits to patients and the US healthcare system, alike. For SLE patients at risk for MLN however, predictive autoantigens are only just now becoming available, with a need for subsequent serological tests. Arkana Laboratories, a leading provider of histopathological and molecular nephrology diagnostics, evaluated a broad range of MLN biopsy samples in an effort to identify autoantigens for MLN that may have the same impact as PLA2R and THSD7A have had on non- lupus MN diagnostics. Using laser capture microdissection, immune complex elution, mass spectrometry, and bioinformatic proteomic analysis, two proteins, Exostosin 1 and Exostosin 2 (EXT1/2), emerged as candidate autoantigens for a subset of MLN. Recently published reports from the Mayo Clinic confirm EXT1/2 as likely autoantigens in MLN. Autoantigenicity was confirmed by staining MLN biopsies for EXT1/2, which exhibited tight co-localization with IgG in the glomerular membrane. EXT1/2 also co-immunoprecipitated with IgG in extracts from MLN kidney biopsy tissue. After evaluating over 80 SLE samples, more than 30% were found to be positive for EXT1/2, with only 1% and 4% exhibiting PLA2R or THSD7A signal, respectively. Interestingly, EXT1/2 circulating autoantibodies have not yet been detected in serological analysis indicating that traditional ELISA- based assays using recombinant proteins to detect autoantibodies may not support an EXT1/2 diagnostic test for MLN. This Phase I program will therefore employ phage display biopanning, an unbiased screening method used to identify high affinity peptide binders, against MLN patient sera and immune complexes from patients with exostosin-positive MLN that likely contain EXT1/2 autoantibodies. Peptides which bind EXT1/2 autoantibodies will be further refined to maximize affinity and specificity for future Phase II testing as diagnostic reagents.

项目概要 多达一半的系统性红斑狼疮 (SLE) 患者会发展为狼疮性肾炎 (LN)，并且 近三分之一的 LN 患者会出现肾衰竭，需要透析或移植。一种 LN， 与非膜性狼疮性肾炎 (MLN) 相比，膜性狼疮性肾炎 (MLN) 表现出相似的临床特征和损伤模式。 狼疮相关膜性肾病（MN）。 MN 和 MLN 主要通过组织病理学诊断 肾活检分析。此外，已在 MN 中鉴定出自身抗原，即 M 型磷脂酶 A2 受体 (PLA2R) 自身抗体存在于近 70% 的病例中，并且含有血小板反应蛋白 1 型结构域 大约 2% 的病例中存在 7A (THSD7A) 自身抗体。鉴于这些之间的紧密联系 自身抗体和 MN，在消除患者肾活检方面取得了重大进展 PLA2R 和 THSD7A 自身抗体血清学阳性。此外，针对特定人群的血清学检测 事实证明，激发性自身抗体（抗 PLA2R 或抗 THSD7A）受到肾脏病学家的高度重视 监测治疗反应并指导患者护理。减少肾活检的要求 精准医疗方法的进展为患者和美国带来了巨大的好处 医疗保健系统，同样如此。然而，对于有 MLN 风险的 SLE 患者，预测性自身抗原才刚刚出现 变得可用，需要进行后续的血清学测试。 Arkana Laboratories，领先的供应商 组织病理学和分子肾病学诊断，评估了广泛的 MLN 活检样本 努力识别 MLN 的自身抗原，这可能与 PLA2R 和 THSD7A 对非 狼疮 MN 诊断。使用激光捕获显微切割、免疫复合物洗脱、质谱分析和 生物信息蛋白质组分析显示，两种蛋白质，外骨蛋白 1 和外骨蛋白 2 (EXT1/2)，成为候选蛋白 MLN 子集的自身抗原。梅奥诊所最近发表的报告证实了 EXT1/2 的可能性 MLN 中存在自身抗原。通过对 MLN 活检组织进行 EXT1/2 染色来证实自身抗原性，该样本表现出紧密的 与肾小球膜中的 IgG 共定位。 EXT1/2 还与提取物中的 IgG 进行免疫共沉淀 来自 MLN 肾活检组织。对 80 多个 SLE 样本进行评估后，发现超过 30% 呈阳性 对于 EXT1/2，分别只有 1% 和 4% 表现出 PLA2R 或 THSD7A 信号。有趣的是，EXT1/2 血清学分析中尚未检测到循环自身抗体，这表明传统的 ELISA- 使用重组蛋白检测自身抗体的检测可能不支持 EXT1/2 诊断测试 对于MLN。因此，该第一阶段计划将采用噬菌体展示生物淘选，这是一种无偏见的筛选方法 用于识别针对 MLN 患者血清和来自患有 MLN 患者的免疫复合物的高亲和力肽结合剂 外骨蛋白阳性 MLN 可能含有 EXT1/2 自身抗体。结合 EXT1/2 自身抗体的肽 将进一步完善，以最大限度地提高未来 II 期测试作为诊断试剂的亲和力和特异性。