Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library
Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library
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DOI：
10.1016/s0022-1759(00)00340-9
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发表时间：
2001-02-01
影响因子：
2.2
通讯作者：
Marks, JD
中科院分区：
文献类型：
--
作者：
Heitner, T;Moor, A;Marks, JD
开发靶向癌症治疗的第一步是产生一种配体，该配体与一种受体结合，该受体要么是肿瘤特异性的，要么是在肿瘤中充分过度表达的，以提供靶向性。在这项工作中，我们制备了抗EGF受体(EGFR)的人源性单抗，单链抗体(ScFv)是通过在高表达EGFR的肿瘤细胞(A431)或转EGFR基因的中国仓鼠卵巢细胞(CHO/EGFR细胞)上筛选噬菌体展示文库并从细胞内回收内吞噬菌体来直接筛选的。从A431细胞和CHO/EGFR细胞上分别分离到3个独特的单链抗体，2个来自A431细胞，2个来自CHO/EGFR细胞。这三种单链抗体都能结合一组肿瘤细胞上表达的天然受体，而不能结合EGFR阴性细胞。共聚焦显微镜显示，表达EGFR的细胞可吞噬由ScFv构建的噬菌体抗体和多价免疫脂质体。天然单链抗体主要染色于细胞表面，细胞内染色较少。结果表明，结合天然细胞表面受体的噬菌体抗体可以直接在高表达的细胞系或转基因细胞上进行选择。使用转基因细胞系可以在不需要蛋白表达和纯化的情况下选择针对天然受体的抗体，显著加快针对基因组序列的靶向抗体的产生。根据使用的形式，抗体可以用来将分子输送到细胞表面或细胞内。(C)2001年爱思唯尔科学公司。版权所有。
The first step in developing a targeted cancer therapeutic is generating a ligand that binds to a receptor which is either tumor specific or sufficiently overexpressed in tumors to provide targeting specificity. For this work, we generated human monoclonal antibodies to the EGF receptor (EGFR), an antigen overexpressed on many solid tumors, single chain Fv (scFv) antibody fragments were directly selected by panning a phage display library on tumor cells (A431) overexpressing EGFR or Chinese hamster ovary cells (CHO/EGFR cells) transfected with the EGFR gene and recovering endocytosed phage from within the cell. Three unique scFvs were isolated, two from selections on A431 cells and two from selections on CHO/EGFR cells. All three scFv bound native receptor as expressed on a panel of tumor cells and did not bind EGFR negative cells. Phage antibodies and multivalent immunoliposomes constructed from scFv were endocytosed by EGFR expressing cells as shown by confocal microscopy. Native scFv primarily stained the cell surface, with less staining intracellularly. The results demonstrate how phage antibodies binding native cell surface receptors can be directly selected on overexpressing cell lines or transfected cells. Use of a transfected cell line allows selection of antibodies to native receptors without the need for protein expression and purification, significantly speeding the generation of targeting antibodies to genomic sequences. Depending upon the format used, the antibodies can be used to deliver molecules to the cell surface or intracellularly. (C) 2001 Elsevier Science BN. All rights reserved.