Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Medical Biotechnology, School of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Immunology. 2020 Jan;159(1):96-108. doi: 10.1111/imm.13129. Epub 2019 Nov 8.
Among various solid tumours, gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Expansion into the peritoneal cavity, which results from dissemination of diffuse cancer cells, is the main cause of mortality in gastric adenocarcinoma patients. Therefore, investigation of putative biomarkers involved in metastasis is prerequisite for GC management. In an effort to discover potential tumour markers associated with peritoneal metastasis of GC, a semi-synthetic human scFv library (Tomlinson I) was used to isolate novel antibody fragments recognizing MKN-45, a poorly differentiated diffuse gastric adenocarcinoma cell line. Four rounds of subtractive selection each consisting of extensive pre-absorption of phage library with NIH-3T3 murine embryonic fibroblasts and AGS (a well-differentiated intestinal gastric adenocarcinoma) cell line were carried out prior to positive selection on MKN-45 target cells. ELISA-based screening of 192 phage-displayed scFv clones indicated 21 high-affinity binders with specific staining of MKN-45 compared with AGS cells. Diversity analysis of the selected phage-scFvs resulted in five distinct sequences with multiple frequency. Further analysis by ELISA and flow cytometry verified three clones that specifically recognized MKN-45 cells. Liquid chromatography-mass spectrometry analysis of the scFv-immunoprecipitated proteins has led to identification of c-Met, HSP90 α and HSP90 β as candidate biomarkers associated with diffuse GC. Immunohistochemistry revealed the capability of purified scFvs to differentiate diffuse and intestinal gastric adenocarcinoma. Taken together, the isolated MKN-45-specific scFv fragments and their cognate antigens would be beneficial in screening and management as well as targeting and therapy of the diffuse gastric adenocarcinoma.
在各种实体肿瘤中，胃癌（GC）是全球癌症相关死亡的主要原因之一。弥漫性癌细胞扩散导致的腹腔扩张是胃腺癌患者死亡的主要原因。因此，研究可能与胃癌转移相关的生物标志物是 GC 管理的前提。为了发现与 GC 腹膜转移相关的潜在肿瘤标志物，我们使用半合成人 scFv 文库（Tomlinson I）分离了识别 MKN-45 的新型抗体片段，MKN-45 是一种低分化弥漫性胃腺癌细胞系。在对 MKN-45 靶细胞进行阳性选择之前，进行了四轮亚选择，每轮亚选择都包括用 NIH-3T3 鼠胚胎成纤维细胞和 AGS（一种分化良好的肠胃腺癌）细胞系进行广泛的预吸收噬菌体文库。对 192 个噬菌体展示 scFv 克隆进行 ELISA 筛选，结果显示有 21 个高亲和力结合物与 AGS 细胞相比，对 MKN-45 具有特异性染色。对所选噬菌体-scFv 的多样性分析产生了 5 个具有多个频率的不同序列。进一步通过 ELISA 和流式细胞术分析，验证了 3 个能够特异性识别 MKN-45 细胞的克隆。scFv 免疫沉淀蛋白的液相色谱-质谱分析导致鉴定出 c-Met、HSP90α 和 HSP90β 作为与弥漫性 GC 相关的候选生物标志物。免疫组织化学显示纯化的 scFv 能够区分弥漫性和肠型胃腺癌。总之，分离的 MKN-45 特异性 scFv 片段及其相应的抗原将有助于弥漫性胃腺癌的筛查和管理，以及靶向治疗。
