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

项目摘要 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)，以及 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)与非狼疮性肾炎相比，表现出相似的临床特征和损害类型。 狼疮相关性膜性肾病(MN)。MN和MLN主要是通过组织病理学诊断的。 肾活检的分析。此外，已在MN中鉴定出具有M型磷脂酶A2的自身抗原 受体(PLA2R)自身抗体存在于近70%的病例中，而凝血酶反应蛋白-1结构域包含 7A(THSD7A)自身抗体存在于大约2%的病例中。鉴于它们之间的紧密联系 自身抗体和MN，在表现为肾活检的患者中有显著的趋势 抗PLA2R和THSD7A自身抗体的血清学阳性。此外，针对该病毒的特异性血清学检测 激发自身抗体(抗PLA2R或抗THSD7A)已被证明是肾病医生高度重视的 监测治疗反应，指导病人护理。减少对肾脏活检的需求并 迈向精准医学方法对患者和美国来说都是巨大的好处 医疗保健系统，类似的。然而，对于系统性红斑狼疮患者来说，预测性自身抗原才刚刚出现。 可以获得，需要进行后续的血清学检测。Arkana实验室，领先的 组织病理学和分子肾病学诊断，评估了广泛的MLN活检样本 努力确定可能与PLA2R和THSD7A具有相同影响的MLN自身抗原对非 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 生物信息学蛋白质组学分析表明，Exostosin 1和Exostosin 2(EXT1/2)是候选蛋白质 MLN子集的自身抗原。梅奥诊所最近发表的报告证实EXT1/2很可能 Mln中的自身抗原。通过对ext1/2的mln活检染色证实自身抗原性。 与免疫球蛋白在肾小球膜上的共定位。EXT1/2还与提取物中的免疫球蛋白免疫共沉淀 取自百万肾活检组织。在评估了80多个系统性红斑狼疮样本后，发现30%以上的样本呈阳性 对于EXT1/2，分别只有1%和4%的人显示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，这表明传统的ELISA- 使用重组蛋白检测自身抗体的基础检测可能不支持EXT1/2诊断测试 为了百万人。因此，这一阶段的计划将采用噬菌体展示生物扫描，这是一种无偏见的筛选方法 用于鉴定抗MLN患者血清和患者免疫复合物的高亲和力多肽结合体 Exostosin阳性的MLN可能含有EXT1/2自身抗体。结合EXT1/2自身抗体的多肽 将进一步改进，以最大限度地提高亲和力和特异性，以便将来作为诊断试剂进行第二阶段测试。

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.