Peptides from the Variable Region of Specific Antibodies Are Shared among Lung Cancer Patients

Late diagnosis of lung cancer is still the main reason for high mortality rates in lung cancer. Lung cancer is a heterogeneous disease which induces an immune response to different tumor antigens. Several methods for searching autoantibodies have been described that are based on known purified antigen panels. The aim of our study is to find evidence that parts of the antigen-binding-domain of antibodies are shared among lung cancer patients. This was investigated by a novel approach based on sequencing antigen-binding-fragments (Fab) of immunoglobulins using proteomic techniques without the need of previously known antigen panels. From serum of 93 participants of the NELSON trial IgG was isolated and subsequently digested into Fab and Fc. Fab was purified from the digested mixture by SDS-PAGE. The Fab containing gel-bands were excised, tryptic digested and measured on a nano-LC-Orbitrap-Mass-spectrometry system. Multivariate analysis of the mass spectrometry data by linear canonical discriminant analysis combined with stepwise logistic regression resulted in a 12-antibody-peptide model which was able to distinguish lung cancer patients from controls in a high risk population with a sensitivity of 84% and specificity of 90%. With our Fab-purification combined Orbitrap-mass-spectrometry approach, we found peptides from the variable-parts of antibodies which are shared among lung cancer patients.

肺癌诊断延迟仍是导致其高死亡率的核心诱因。肺癌属于异质性疾病，可针对不同肿瘤抗原引发免疫应答。目前已有多项基于已知纯化抗原组的自身抗体筛选方法被报道。本研究旨在寻找肺癌患者体内抗体的抗原结合域存在共有序列的相关证据。为此，我们采用一种全新的研究策略：无需预先构建已知抗原组，而是借助蛋白质组学技术对免疫球蛋白的抗原结合片段（Fab）进行测序分析。研究从NELSON试验的93名受试者血清中分离出免疫球蛋白G（IgG），随后将其酶解为Fab段与Fc段；通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）从酶解混合液中纯化得到Fab段。切下含有Fab段的凝胶条带，经胰蛋白酶酶解后，使用纳流液相色谱-轨道阱质谱系统进行检测。通过线性典型判别分析结合逐步逻辑回归对质谱数据开展多变量分析，最终构建出包含12种抗体肽段的预测模型；该模型可在高危人群中有效区分肺癌患者与健康对照，灵敏度达84%，特异度达90%。本研究通过Fab纯化结合轨道阱质谱的分析方法，成功发现了肺癌患者体内共有的抗体可变区肽段。