Table_1_Human-murine chimeric autoantibodies with high affinity and specificity for systemic sclerosis.xlsx

Scleroderma 70 (Scl-70) is commonly used in the clinic for aiding systemic sclerosis (SSc) diagnosis due to its recognition as autoantibodies in the serum of SSc patients. However, obtaining sera positive for anti-Scl-70 antibody can be challenging; therefore, there is an urgent need to develop a specific, sensitive, and easily available reference for SSc diagnosis. In this study, murine-sourced scFv library were screened by phage display technology against human Scl-70, and the scFvs with high affinity were constructed into humanized antibodies for clinical application. Finally, ten high-affinity scFv fragments were obtained. Three fragments (2A, 2AB, and 2HD) were select for humanization. The physicochemical properties of the amino acid sequence, three-dimensional structural basis, and electrostatic potential distribution of the protein surface of different scFv fragments revealed differences in the electrostatic potential of their CDR regions determined their affinity for Scl-70 and expression. Notably, the specificity test showed the half-maximal effective concentration values of the three humanized antibodies were lower than that of positive patient serum. Moreover, these humanized antibodies showed high specificity for Scl-70 in diagnostic immunoassays for ANA. Among these three antibodies, 2A exhibited most positive electrostatic potential on the surface of the CDRs and highest affinity and specificity for Scl-70 but with least expression level; thus, it may provide new foundations for developing enhanced diagnostic strategies for SSc.

硬皮病70（Scl-70）可识别系统性硬化症（SSc）患者血清中的自身抗体，临床常用于辅助系统性硬化症的诊断。然而，获取抗Scl-70抗体阳性血清颇具挑战，因此亟需开发一种特异性优异、灵敏度良好且易于获取的SSc诊断参照试剂。本研究采用噬菌体展示技术，以人源Scl-70为靶标对鼠源单链可变片段（scFv）文库进行筛选，将获得的高亲和力scFv构建为人源化抗体以推进临床应用。最终本研究共得到10株高亲和力scFv片段，选取其中3株（2A、2AB及2HD）开展人源化改造。通过分析不同scFv片段的氨基酸序列理化性质、三维结构基础及蛋白质表面静电势分布，发现其互补决定区（CDR）的静电势差异，是决定各片段与Scl-70亲和力及表达水平的关键因素。特异性实验结果显示，3株人源化抗体的半数有效浓度（half-maximal effective concentration）均低于阳性患者血清。此外，在抗核抗体（ANA）诊断免疫测定中，上述人源化抗体对Scl-70均表现出极高的特异性。在这3株抗体中，2A的互补决定区表面正静电势最强，对Scl-70的亲和力与特异性最高，但表达水平最低，因此可为开发更优化的SSc诊断策略提供新的研究基础。