Tagliabue E, Centis F, Campiglio M, Mastroianni A, Martignone S, Pellegrini R, Casalini P, Lanzi C, Ménard S, Colnaghi M I
Division of Experimental Oncology E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Int J Cancer. 1991 Apr 1;47(6):933-7. doi: 10.1002/ijc.2910470625.
In order to obtain further information on the biological role of the HER2/neu oncoprotein monoclonal antibodies (MAbs) were produced against the p185 extracellular domain. To immunize the mice and screen the hybridoma supernatants we selected a lung adenocarcinoma cell line (Calu-3), which demonstrated an over-expression of p185HER2 measured as the reactivity with polyclonal rabbit serum to the 14-amino-acid carboxy-terminal-HER2/neu. Two MAbs, designated MGR2 (IgG1) and MGR3 (IgG2), selected for reactivity on Calu-3 and negativity on A43I live cells, the reference target cell for EGF receptor expression, were found to immunoprecipitate a 185-kDa molecule. Immunodepletion experiments with the polyclonal antiserum and cross-competition experiments indicated that the 2 reagents recognized 2 different epitopes located on the p185HER2 molecule. One of the 2 MAbs, MGR3, was found to internalize, induce p185HER2 phosphorylation and inhibit tumor cell growth in vitro. These results indicate that MGR3 is directed against a determinant located in the p185HER2 ligand binding site and may compete with the p185HER2 ligand, but is incapable of inducing a complete mitotic signal.
为了获取关于HER2/neu癌蛋白生物学作用的更多信息，制备了针对p185细胞外结构域的单克隆抗体（MAbs）。为了免疫小鼠并筛选杂交瘤上清液，我们选择了一种肺腺癌细胞系（Calu-3），该细胞系显示p185HER2过表达，通过与多克隆兔血清对14个氨基酸的羧基末端-HER2/neu的反应性来衡量。选择了两种单克隆抗体，命名为MGR2（IgG1）和MGR3（IgG2），它们对Calu-3有反应性，对作为表皮生长因子受体表达参考靶细胞的A431活细胞无反应性，发现它们能免疫沉淀一个185 kDa的分子。用多克隆抗血清进行的免疫耗竭实验和交叉竞争实验表明，这两种试剂识别位于p185HER2分子上的两个不同表位。发现这两种单克隆抗体之一MGR3能内化、诱导p185HER2磷酸化并在体外抑制肿瘤细胞生长。这些结果表明，MGR3针对的是位于p185HER2配体结合位点的一个决定簇，可能与p185HER2配体竞争，但不能诱导完整的有丝分裂信号。
