Younesi Vahid, Nejatollahi Foroogh
Int Immunopharmacol. 2014 Dec;23(2):624-32. doi: 10.1016/j.intimp.2014.10.015.
Overexpression of IL-25 receptor (IL-25R) on the surface of malignant, but not normal, breast epithelial cells contributes to tumorigenic potential of breast cancer cells. IL-25/IL-25R binding results in specific apoptosis in breast cancer cells. In this study, the inhibitory effects of anti-IL-25R scFv antibodies on three breast cancer cells were evaluated. Panning process was performed on a phage library to isolate scFvs against two specific epitopes of IL25R. The reactivities of scFvs were assessed using phage ELISA. Cell binding capacity of the selected scFvs on breast cancer cells was analyzed using flow cytometry. Anti-proliferative and apoptotic effects of scFvs were evaluated by MTT, Annexin V/PI and quantitative caspase 3 activity assays. Two scFvs with frequencies of 47% and 68% were selected against peptides I and II, respectively. Phage ELISA demonstrated the specific reactions of the scFvs against the corresponding peptides. The scFvs against peptides I and II bound to 14% and 21% of MDA-231, 28% and 32% of MCF7, and 21% and 30% of SKBR3 cells, respectively. Treatment of the breast cancer cells with IL-25 and two anti-IL-25R scFvs resulted in significant inhibition of the cell growth in these cell compared to that in IL-25R negative Raji cells. The results also demonstrated 66% and 70% apoptosis cell death and 15.5 and 17 fold increases in caspase- 3 activity in SKBR3 cells induced by scFvs I and II, respectively. Our findings suggest the targeting of IL-25R by scFvs as a new effective strategy in breast cancer immunotherapy.
白细胞介素-25受体（IL-25R）在恶性而非正常乳腺上皮细胞表面的过表达有助于乳腺癌细胞的致瘤潜力。IL-25/IL-25R结合导致乳腺癌细胞发生特异性凋亡。在本研究中，评估了抗IL-25R单链抗体片段（scFv）对三种乳腺癌细胞的抑制作用。在噬菌体文库上进行淘选过程，以分离针对IL25R两个特定表位的scFv。使用噬菌体酶联免疫吸附测定（ELISA）评估scFv的反应性。使用流式细胞术分析所选scFv对乳腺癌细胞的细胞结合能力。通过MTT、膜联蛋白V/碘化丙啶（Annexin V/PI）和定量半胱天冬酶3活性测定评估scFv的抗增殖和凋亡作用。分别针对肽I和肽II选择了频率为47%和68%的两种scFv。噬菌体ELISA证明scFv与相应肽发生特异性反应。针对肽I和肽II的scFv分别与14%和21%的MDA-231细胞、28%和32%的MCF7细胞以及21%和30%的SKBR3细胞结合。与IL-25R阴性的拉吉（Raji）细胞相比，用IL-25和两种抗IL-25R scFv处理乳腺癌细胞导致这些细胞中的细胞生长受到显著抑制。结果还表明，scFv I和scFv II分别诱导SKBR3细胞发生66%和70%的凋亡细胞死亡，并且半胱天冬酶-3活性分别增加15.5倍和17倍。我们的研究结果表明，scFv靶向IL-25R是乳腺癌免疫治疗中的一种新的有效策略。
