Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells
Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells
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DOI：
10.1080/21691401.2018.1478846
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发表时间：
2018-01-01
影响因子：
5.8
通讯作者：
Omidi, Yadollah
中科院分区：
文献类型：
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作者：
Khajeh, Shirin;Tohidkia, Mohammad Reza;Omidi, Yadollah
甘氨酸延伸的胃泌素17（Glycine-extended gastrin 17，G17-Gly）是胃泌素基因的主要加工中间产物，与结直肠癌的发生和发展密切相关。因此，通过抗体实体中和G17-Gly活性可以为CRC患者提供潜在的治疗策略。为此，我们通过针对G17-Gly的不同表位的生物淘选从噬菌体抗体库中分离出全人抗体片段，以获得尽可能高的抗体多样性。ELISA筛选和序列分析鉴定了2个单链抗体和4个V-L抗体片段。通过SPR的抗体片段的动力学分析揭示K-D值在纳摩尔范围内（87.9- 334 nM）。在CRC细胞系HCT-116中分析所选抗G17-Gly抗体片段的生长抑制和凋亡测定，HCT-116被充分表征为表达胃泌素中间物质但不表达酰胺化胃泌素。抗体片段对HCT-116细胞增殖表现出显着抑制作用，范围为对照组的36.5%至73%。此外，Annexin V/PI染色表明scFv H8和V-LG 8处理的细胞的凋亡率分别为45.8%和63%。基于这些结果，我们首次证明了抗G17-Gly人scFv和V-L抗体的分离在G17-Gly应答性肿瘤中具有潜在的治疗应用。
Glycine-extended gastrin 17 (G17-Gly), a dominant processing intermediate of gastrin gene, has been implicated in the development or maintenance of colorectal cancers (CRCs). Hence, neutralizing G17-Gly activity by antibody entities can provide a potential therapeutic strategy in the patients with CRCs. To this end, we isolated fully human antibody fragments from a phage antibody library through biopanning against different epitopes of G17-Gly in order to obtain the highest possible antibody diversity. ELISA screening and sequence analysis identified 2 scFvs and 4 V-L antibody fragments. Kinetic analysis of the antibody fragments by SPR revealed K-D values to be in the nanomolar range (87.9-334nM). The selected anti-G17-Gly antibody fragments were analyzed for growth inhibition and apoptotic assays in a CRC cell line, HCT-116, which is well-characterized for expressing gastrin intermediate species but not amidated gastrin. The antibody fragments exhibited significant inhibition of HCT-116 cells proliferation ranging from 36.5 to 73% of controls. Further, Annexin V/PI staining indicated that apoptosis rates of scFv H8 and V-L G8 treated cells were 45.8 and 63%, respectively. Based on these results, we for the first time, demonstrated the isolation of anti-G17-Gly human scFv and V-L antibodies with potential therapeutic applications in G17-Gly-responsive tumors.