表位特异性决定抗血小板衍生生长因子受体 α 自身抗体在系统性硬化症中的致病性和可检测性。

Epitope Specificity Determines Pathogenicity and Detectability of Anti-Platelet-Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis.

机构信息

Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.

Università di Torino, Turin, Italy.

出版信息

Arthritis Rheumatol. 2015 Jul;67(7):1891-903. doi: 10.1002/art.39125.

DOI:10.1002/art.39125

PMID:25808833

Abstract

OBJECTIVE

To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor α (PDGFRα) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFRα autoantibodies.

METHODS

Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRα and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRα IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRα recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRα extracellular domains, and expression analyses of alanine-scanned PDGFRα mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRα autoantibodies or, selectively, the agonistic ones.

RESULTS

Three types of anti-PDGFRα recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VH PAM-Vκ 13B8 recognized 1 linear epitope, whereas agonistic VH PAM-Vλ 16F4 and VH PAM-Vκ 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRα antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum VH PAM-Vκ 16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the VH PAM-Vκ 16F4 epitope inhibited PDGFRα signaling triggered by serum IgG from SSc patients.

CONCLUSION

Agonistic anti-PDGFRα autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRα signaling in patients with SSc.

摘要

目的

鉴定针对血小板衍生生长因子受体 α（PDGFRα）的自身抗体在系统性硬化症（SSc）中识别的表位，并开发用于检测血清抗 PDGFRα 自身抗体的新型检测方法。

方法

从 1 名 SSc 患者（命名为 PAM）的 EBV 永生化 B 细胞中筛选出与 PDGFRα 结合并诱导成纤维细胞中产生活性氧的 IgG。将抗 PDGFRα IgG 的可变区克隆到 IgG 表达载体中，以生成不同的重组人单克隆自身抗体（mAb），并通过结合和功能测定对其进行表征。通过分子对接、表面等离子体共振结合测定、跨越 PDGFRα 细胞外结构域的构象肽文库筛选以及 PDGFRα 突变体的表达分析来定义抗 PDGFRα 重组人 mAb 的表位。建立直接或竞争性酶联免疫吸附测定法来检测所有血清抗 PDGFRα 自身抗体或选择性地检测激动性自身抗体。

结果

生成了 3 种具有相同 VH 但不同 VL 链的抗 PDGFRα 重组人 mAb。非激动性 VH PAM-Vκ 13B8 识别 1 个线性表位，而激动性 VH PAM-Vλ 16F4 和 VH PAM-Vκ 16F4 识别 2 个不同的构象表位。在 70 名 SSc 患者中的 66 名、130 名健康对照者中的 63 名、26 名原发性雷诺现象（RP）患者中的 11 名和 29 名系统性红斑狼疮（SLE）患者中的 13 名中检测到血清抗 PDGFRα 抗体。在 34 名 SSc 患者中的 24 名中发现了与血清 VH PAM-Vκ 16F4 相似的抗体，但在健康对照者、原发性 RP 患者或 SLE 患者中未发现。组成 VH PAM-Vκ 16F4 表位的肽可抑制 SSc 患者血清 IgG 触发的 PDGFRα 信号转导。