1 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
2 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
SLAS Discov. 2017 Apr;22(4):408-417. doi: 10.1177/2472555216689659. Epub 2017 Jan 31.
Receptor tyrosine kinase-like orphan receptor (ROR) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including cell survival, differentiation, cell migration, cell communication, cell polarity, proliferation, metabolism, and angiogenesis. ROR1 has recently been shown to be expressed in various types of cancer cells but not normal cells. Pharmacokinetics and pharmacodynamics of single-chain Fragment variable (scFv) antibodies provide potential therapeutic advantages over whole antibody molecules. In the present study, scFvs against a specific peptide from the extracellular domain of ROR1 were selected using phage display technology. The selected scFvs were further characterized using polyclonal and monoclonal phage enzyme-linked immunosorbent assay (ELISA), soluble monoclonal ELISA, colony PCR, and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies were also evaluated in lymphoma and myeloma cancer cell lines using MTT and annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the ROR1 peptide. Colony PCR confirmed the presence of full-length V and Vκ inserts. The percentages of cell growth after 24 h of treatment of cells with individual scFv revealed that the scFv significantly inhibited the growth of the RPMI8226 and chronic lymphocytic leukemia (CLL) cells in comparison with the untreated cells ( p < 0.05). Interestingly, 24-h treatment with specific scFv induced apoptosis cell death in the RPMI8226 and CLL cells. Taken together, our results demonstrate that targeting of ROR1 using peptide-specific scFv can be an effective immunotherapy strategy in hematological malignancies.
受体酪氨酸激酶样孤儿受体（ROR）蛋白是一类保守的酪氨酸激酶受体家族，在细胞存活、分化、细胞迁移、细胞通讯、细胞极性、增殖、代谢和血管生成等发育过程中发挥作用。最近的研究表明，ROR1 在各种类型的癌细胞中表达，但在正常细胞中不表达。单链片段可变（scFv）抗体的药代动力学和药效学为治疗提供了潜在的优势，优于全抗体分子。在本研究中，使用噬菌体展示技术从 ROR1 细胞外结构域的特定肽段中筛选出 scFv。通过多克隆和单克隆噬菌体酶联免疫吸附试验（ELISA）、可溶性单克隆 ELISA、菌落 PCR 和测序进一步对筛选出的 scFv 进行了鉴定。通过 MTT 和 Annexin V/PI 测定法，还评估了选定的 scFv 抗体在淋巴瘤和骨髓瘤癌细胞系中的抗增殖和凋亡作用。ELISA 结果表明，分离的 scFv 与 ROR1 肽特异性反应。菌落 PCR 证实了全长 V 和 Vκ 插入物的存在。单独用 scFv 处理细胞 24 小时后细胞生长的百分比表明，与未经处理的细胞相比，scFv 显著抑制了 RPMI8226 和慢性淋巴细胞白血病（CLL）细胞的生长（p<0.05）。有趣的是，特异性 scFv 治疗 24 小时后可诱导 RPMI8226 和 CLL 细胞凋亡。综上所述，我们的结果表明，使用肽特异性 scFv 靶向 ROR1 可能是血液恶性肿瘤的一种有效的免疫治疗策略。
