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 多达一半的全身性红斑红肿患者（SLE）会出现狼疮肾炎（LN），并且 几乎三分之一的LN患者会出现肾脏衰竭，需要透析或移植。一种LN， 与非非 - 狼疮相关的膜肾病（MN）。 MN和MLN在很大程度上通过组织病理学诊断 分析肾脏活检。此外，已在MN中鉴定出自动抗原，并用M型磷脂酶A2鉴定 在近70％的病例中存在的受体（PLA2R）自身抗体和含有血小板素的1型结构域 7A（THSD7A）自身抗体大约有2％的病例。考虑到这些之间的紧密联系 自身抗体和MN，在表现出的患者中消除肾脏活检有重大运动 针对PLA2R和THSD7A的自身抗体的血清学阳性。此外，针对特定的血清学测试 事实证明 监视对治疗和指导患者护理的反应。减少对肾脏活检和 朝着精确的医学方法迈进，对患者和美国代表了巨大的好处 医疗保健系统，都一样。但是，对于有MLN风险的SLE患者，预测性自动抗原刚才现在 可用，需要随后的血清学检查。 Arkana Laboratories，领先的提供商 组织病理学和分子肾脏病诊断，评估了广泛的MLN活检样品 努力确定MLN的自动抗原，可能与PLA2R和THSD7A对非 - 的影响相同 狼疮MN诊断。使用激光捕获显微解剖，免疫复合洗脱，质谱法和 生化蛋白质组学分析，两种蛋白质，外生蛋白1和Exostosin 2（Ext1/2），作为候选者出现 MLN子集的自动抗原。最近发表的Mayo诊所的报告确认Ext1/2的可能性很大 MLN中的自动抗原。通过对Ext1/2的MLN活检染色，可以证实自身抗原性，该活检表现为紧密 与肾小球膜中的IgG共定位。 Ext1/2还与提取物中的IgG共免疫沉淀 来自MLN肾脏活检组织。评估超过80个SLE样品后，发现超过30％为正 对于Ext1/2，只有1％和4％的pla2r或thsd7a信号。有趣的是，Ext1/2 在血清学分析中尚未检测到循环自身抗体，表明传统的Elisa- 使用重组蛋白检测自身抗体的基于基于的测定可能不支持EXT1/2诊断测试 用于MLN。因此，该阶段I计划将采用噬菌体显示生物扫描，这是一种公正的筛选方法 用于鉴定高亲和肽粘合剂，以针对MLN患者血清和来自患者的免疫复合物 可能包含Ext1/2自身抗体的外蛋白阳性MLN。结合Ext1/2自动抗体的肽 将进一步完善，以最大化对未来II期测试作为诊断试剂的亲和力和特异性。