Recombinant Human IgG Antibodies Recognizing Distinct Extracellular Domains of EGF Receptor Exhibit Different Degrees of Growth Inhibitory Effects On Human A431 Cancer Cells.
Recombinant Human IgG Antibodies Recognizing Distinct Extracellular Domains of EGF Receptor Exhibit Different Degrees of Growth Inhibitory Effects On Human A431 Cancer Cells.
复制标题
识别 EGF 受体不同胞外域的重组人 IgG 抗体对人 A431 癌细胞表现出不同程度的生长抑制作用。
DOI：
10.1016/j.yexcr.2013.03.002
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发表时间：
2013
影响因子：
3.7
通讯作者：
N.
中科院分区：
文献类型：
--
作者：
Chang;C.;Takayanagi;A.;Yoshida;T.;Shimizu;N.
最近，我们通过两种靶向邻近标记方法筛选Keio噬菌体展示单链抗体库，分离到4种不同的抗人表皮生长因子受体（EGFR）单链抗体。在本研究中，这些单价scFv抗体通过重组技术使用特别设计的表达载体转化为人源化形式（hIgG）的二价IgG，随后在CHO细胞中产生蛋白质。使用表达EGFR缺失突变体的几种不同转化的人BJ细胞系检查所得重组hIgG的结合特异性，每种细胞系缺乏4种不同胞外结构域（L1、L2、C1和C2）之一。免疫荧光显微镜和免疫沉淀试验表明，4种不同的hIgG分别与3个不同的结构域（L1、C1和C2）结合。然后，进一步检查这些hIgG对过表达EGFR的人A431癌细胞的生物学效应。结果表明，与L1结合的hIgG38和与C2结合的hIgG45基本上抑制了EGF诱导的EGFR磷酸化，导致A431癌细胞的生长抑制。相反，与C1结合的hIgG40和与C2的另一个位点（表位）结合的hIgG42没有表现出这样的抑制作用。因此，新产生的四种重组hIgG抗体识别EGFR的3个不同胞外结构域中的4个不同位点（表位），并对癌细胞表现出不同的生物学效应。这些特征与目前使用的治疗性抗EGFR抗体有些不同。因此，这些hIgG抗体作为EGFR介导的信号转导机制的详细分子分析的研究工具将是非常宝贵的，更重要的是作为治疗某些类型癌症的新治疗剂的可能应用。
Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.