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.

项目摘要 高达一半的系统性红斑狼疮（SLE）患者会发展为狼疮性肾炎（LN）， 将近三分之一的LN患者将发展为需要透析或移植的肾衰竭。一种类型的LN， 膜性狼疮性肾炎（MLN）与非膜性狼疮性肾炎相比，表现出相似的临床特征和损伤模式。 狼疮相关性膜性肾病（MN）。MN和MLN主要通过组织病理学诊断 肾活检的分析。此外，已在MN中鉴定出自身抗原，其中M型磷脂酶A2 近70%的病例中存在PLA 2 R自身抗体， 7A（THSD 7A）自身抗体存在于大约2%的病例中。考虑到这两者之间的紧密联系 自身抗体和MN，有显着的运动，消除肾活检的患者， 针对PLA 2 R和THSD 7A的自身抗体的血清学阳性。此外，血清学试验的具体为 激发性自身抗体（抗PLA 2 R或抗THSD 7A）已被肾脏病学家高度重视， 监测对治疗的反应并指导患者护理。减少对肾活检的要求， 向精确医学方向发展对患者和美国都有巨大的好处 医疗保健系统也一样。然而，对于有MLN风险的SLE患者，预测性自身抗原只是刚刚开始， 需要随后进行血清学检测。Arkana实验室是一家领先的 组织病理学和分子肾脏学诊断，评估了广泛的MLN活检样本， 努力鉴定可能与PLA 2 R和THSD 7A具有相同影响的MLN自身抗原， 狼疮MN诊断。使用激光捕获显微切割，免疫复合物洗脱，质谱， 生物信息学蛋白质组学分析显示，Exostosin 1和Exostosin 2（EXT 1/2）两个蛋白质成为候选蛋白 MLN亚群的自身抗原。最近发表的来自马约诊所的报告证实了EXT 1/2可能 MLN中的自身抗原。通过对MLN活检组织的EXT 1/2染色证实了自身抗原性， 在肾小球膜中与IgG共定位。EXT 1/2也与提取物中的IgG共免疫沉淀 来自MLN肾活检组织。对80余例SLE患者进行检测，阳性率超过30% 对于EXT 1/2，仅1%和4%分别显示PLA 2 R或THSD 7A信号。有趣的是，EXT 1/2 在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基于检测试剂盒可能不支持EXT 1/2诊断试验 关于MLN因此，该第一阶段计划将采用噬菌体展示生物淘选，一种无偏筛选方法 用于鉴定针对MLN患者血清和来自MLN患者的免疫复合物的高亲和力肽结合剂， 可能含有EXT 1/2自身抗体的外生软骨素阳性MLN。结合EXT 1/2自身抗体的肽 将进一步完善，以最大限度地提高亲和力和特异性，为未来的第二阶段测试作为诊断试剂。