Nejatollahi Foroogh, Bayat Payam, Moazen Bahareh
Shiraz HIV/AIDS research center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran.
Recombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran.
F1000Res. 2017 Feb 17;6:156. doi: 10.12688/f1000research.10803.1. eCollection 2017.
Single chain antibody (scFv) has shown interesting results in cancer immunotargeting approaches, due to its advantages over monoclonal antibodies. Regeneration and tolerance factor (RTF) is one of the most important regulators of extracellular and intracellular pH in eukaryotic cells. In this study, the inhibitory effects of a specific anti-RTF scFv were investigated and compared between three types of prostate cancer and two types of glioblastoma cells. A phage antibody display library of scFv was used to select specific scFvs against RTF using panning process. The reactivity of a selected scFv was assessed by phage ELISA. The anti-proliferative and apoptotic effects of the antibody on prostate cancer (PC-3, Du-145 and LNCaP) and glioblastoma (U-87 MG and A-172) cell lines were investigated by MTT and Annexin V/PI assays. A specific scFv with frequency 35% was selected against RTF epitope. This significantly inhibited the proliferation of the prostate cells after 24 h. The percentages of cell viability (using 1000 scFv/cell) were 52, 61 and 73% for PC-3, Du-145 and LNCaP cells, respectively, compared to untreated cells. The antibody (1000 scFv/cell) induced apoptosis at 50, 40 and 25% in PC-3, Du-145 and LNCaP cells, respectively. No growth inhibition and apoptotic induction was detected for U-87 and A172 glioblastoma cells. Anti-RTFscFv significantly reduced the proliferation of the prostate cancer cells. The inhibition of cell growth and apoptotic induction effects in PC-3 cells were greater than Du-145 and LNCaP cells. This might be due to higher expression of RTF antigen in PC-3 cells and/or better accessibility of RTF to scFv antibody. The resistance of glioblastoma cells to anti-RTF scFv offers the existence of mechanism(s) that abrogate the inhibitory effect(s) of the antibody to RTF. The results suggest that the selected anti-RTF scFv antibody could be an effective new alternative for prostate cancer immunotherapy.
单链抗体（scFv）由于其相对于单克隆抗体的优势，在癌症免疫靶向治疗方法中已显示出有趣的结果。再生和耐受因子（RTF）是真核细胞中细胞外和细胞内pH值最重要的调节因子之一。在本研究中，研究并比较了一种特异性抗RTF scFv对三种前列腺癌细胞和两种胶质母细胞瘤细胞的抑制作用。使用scFv的噬菌体抗体展示文库通过淘选过程筛选针对RTF的特异性scFv。通过噬菌体ELISA评估所选scFv的反应性。通过MTT和Annexin V/PI测定法研究该抗体对前列腺癌（PC-3、Du-145和LNCaP）和胶质母细胞瘤（U-87 MG和A-172）细胞系的抗增殖和凋亡作用。针对RTF表位筛选出频率为35%的特异性scFv。这在24小时后显著抑制了前列腺细胞的增殖。与未处理的细胞相比，PC-3、Du-145和LNCaP细胞的细胞活力百分比（使用1000 scFv/细胞）分别为52%、61%和73%。该抗体（1000 scFv/细胞）在PC-3、Du-145和LNCaP细胞中分别诱导50%、40%和25%的细胞凋亡。未检测到U-87和A172胶质母细胞瘤细胞有生长抑制和凋亡诱导现象。抗RTF scFv显著降低了前列腺癌细胞的增殖。PC-3细胞中的细胞生长抑制和凋亡诱导作用大于Du-145和LNCaP细胞。这可能是由于PC-3细胞中RTF抗原的表达较高和/或RTF对scFv抗体的可及性更好。胶质母细胞瘤细胞对抗RTF scFv的抗性表明存在消除该抗体对RTF抑制作用的机制。结果表明，所选的抗RTF scFv抗体可能是前列腺癌免疫治疗的一种有效的新选择。
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