Kazemi Tohid, Tahmasebi Fathollah, Bayat Ali Ahmad, Mohajer Neda, Khoshnoodi Jalal, Jeddi-Tehrani Mahmood, Rabbani Hodjatallah, Shokri Fazel
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Enghelab Ave., Tehran, Iran.
Hybridoma (Larchmt). 2011 Aug;30(4):347-53. doi: 10.1089/hyb.2011.0023.
HER2 proto-oncogene encodes a transmembrane receptor tyrosine kinase overexpressed in a variety of solid tumors. Several mouse monoclonal antibodies (MAbs) have been developed that recognize the extracellular part of HER2; of them two MAbs were humanized and employed for targeted immunotherapy. In this study we aimed to produce murine MAbs that specifically recognize the extracellular domain of human HER2. BALB/c mice were first primed with HER2-transfected NIH-3T3 cells and then boosted with recombinant extracellular part of HER2. Splenocytes from hyperimmunized mice were fused with myeloma cells and growing hybridomas were selected and screened for HER2 reactivity by an indirect ELISA. HER2-specific hybridomas were selected, cloned by limiting dilution assay, and further characterized by Western blotting and flow cytometry techniques. All clones showed positive reactivity to HER2 with binding affinity, ranging from 1.9×10(8) to 5×10(9), and stained HER2-transfected cells and malignant cells overexpressing HER2. None of the MAbs inhibited the binding of trastuzumab (Herceptin(®)) to HER2, indicating recognition of distinct epitopes by these MAbs. Based on these findings, our MAbs could be potentially used for selective targeting of HER2-expressing malignancies.
HER2原癌基因编码一种在多种实体瘤中过表达的跨膜受体酪氨酸激酶。已经开发出几种识别HER2细胞外部分的小鼠单克隆抗体（MAb）；其中两种MAb被人源化并用于靶向免疫治疗。在本研究中，我们旨在制备特异性识别人类HER2细胞外结构域的鼠源MAb。首先用HER2转染的NIH-3T3细胞对BALB/c小鼠进行免疫，然后用HER2的重组细胞外部分进行加强免疫。将超免疫小鼠的脾细胞与骨髓瘤细胞融合，选择生长的杂交瘤，并通过间接ELISA筛选其对HER2的反应性。选择HER2特异性杂交瘤，通过有限稀释法进行克隆，并通过蛋白质印迹和流式细胞术技术进一步鉴定。所有克隆对HER2均表现出阳性反应，结合亲和力范围为1.9×10(8)至5×10(9)，并对HER2转染细胞和过表达HER2的恶性细胞进行染色。没有一种MAb抑制曲妥珠单抗（赫赛汀(®)）与HER2的结合，表明这些MAb识别不同的表位。基于这些发现，我们的MAb可能潜在地用于选择性靶向表达HER2的恶性肿瘤。
