Ahmadzadeh Maryam, Mohit Elham
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, No. 2660, Vali-e-Asr Ave, Tehran, 1991953381, Iran.
Food and Drug Administration, The Ministry of Health and Medical Education, Tehran, Iran.
Biotechnol Lett. 2023 Mar;45(3):371-385. doi: 10.1007/s10529-022-03342-y. Epub 2023 Jan 18.
Interferon-γ-inducible protein 10 (IP-10) is a potent antitumor agent and acts by its angiostatic and immunomodulatory properties. IP-10 can target to tumor site by linking with single chain variable fragment (scFv) that recognized specific tumor antigen. In this study, we evaluated biological activity of the fusion protein including IP-10 and anti-HER2 scFv (IP-10-(anti-HER2 scFv)).
The HER2- and cell-based ELISA as well as the flow cytometry analysis demonstrated that the fusion protein specifically binds to HER2 antigen. In addition, competitive ELISA demonstrated that the fusion protein recognized the same epitope of HER2 antigen as trastuzumab. The results of MTT assay demonstrated that the growth of HER2-enriched SK-BR3 cells was inhibited in the presence of the fusion protein. Moreover, the cytotoxic effect of the fusion protein was not significantly different from that of trastuzumab. However, no significant cytotoxic effect compared to trastuzumab and anti-HER2 scFv was observed in HER2-low-expressing MDA-MB-231 cells. The obtained findings demonstrated that IP-10-(anti-HER2 scFv) can selectively reduce the cell viability in HER2 cells. Moreover, similar inhibitory effect on growth of both SK-BR-3 and MDA-MB-231 cell lines was observed in the presence of anti-HER2 scFv protein even at high concentration after 72 h. The chemotaxis properties of the fusion protein were also analyzed by a chemotaxis assay. It was demonstrated that the fusion protein induced migration of activated T cell similar to recombinant IP-10 protein.
Our findings suggested that IP-10-(anti-HER2 scFv) fusion protein can specifically direct IP-10 to the HER2-expressing tumor cells and may act as an adjuvant along with HER2-based vaccine to gather the elicited immune response at the site of HER2-overexpressimg tumors.
干扰素-γ诱导蛋白10（IP-10）是一种有效的抗肿瘤药物，通过其血管生成抑制和免疫调节特性发挥作用。IP-10可通过与识别特定肿瘤抗原的单链可变片段（scFv）连接而靶向肿瘤部位。在本研究中，我们评估了包含IP-10和抗HER2 scFv的融合蛋白（IP-10-(抗HER2 scFv)）的生物学活性。
基于HER2和细胞的ELISA以及流式细胞术分析表明，融合蛋白特异性结合HER2抗原。此外，竞争性ELISA表明，融合蛋白识别的HER2抗原表位与曲妥珠单抗相同。MTT试验结果表明，在融合蛋白存在的情况下，富含HER2的SK-BR3细胞的生长受到抑制。此外，融合蛋白的细胞毒性作用与曲妥珠单抗无显著差异。然而，在HER2低表达的MDA-MB-231细胞中，未观察到与曲妥珠单抗和抗HER2 scFv相比有显著的细胞毒性作用。所获得的结果表明，IP-10-(抗HER2 scFv)可选择性降低HER2细胞中的细胞活力。此外，即使在72小时后高浓度的抗HER2 scFv蛋白存在的情况下，在SK-BR-3和MDA-MB-231细胞系中均观察到对生长的类似抑制作用。还通过趋化试验分析了融合蛋白的趋化特性。结果表明，融合蛋白诱导活化T细胞迁移的作用与重组IP-10蛋白相似。
我们的研究结果表明，IP-10-(抗HER2 scFv)融合蛋白可将IP-10特异性导向表达HER2的肿瘤细胞，并可能作为基于HER2的疫苗的佐剂，在HER2过表达肿瘤部位聚集引发的免疫反应。
