Zhou Yu, Marks James D
Department of Anesthesia and Perioperative Care, University of California, San Francisco, California, USA.
Methods Enzymol. 2012;502:43-66. doi: 10.1016/B978-0-12-416039-2.00003-3.
Phage antibody technology can be used to generate human antibodies to essentially any antigen. Many therapeutic target antigens are cell surface receptors, which can be challenging targets for antibody generation. In addition, for many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but also are internalized into the cell upon binding. This allows use of the antibody to deliver a range of payloads into the cell to achieve a therapeutic effect. In this chapter, we describe how human phage antibody libraries can be selected directly on tumor cell lines to generate antibodies that bind cell surface receptors and which upon binding are rapidly internalized into the cell. Specific protocols show how to (1) directly select cell binding and internalizing antibodies from human phage antibody libraries, (2) screen the phage antibodies in a high-throughput flow cytometry assay for binding to the tumor cell line used for selection, (3) identify the antigen bound by the phage antibody using immunoprecipitation and mass spectrometry, and (4) direct cell binding and internalizing selections to a specific tumor antigen by sequential selection on a tumor cell line followed by selection on yeast displaying the target tumor antigen on the yeast surface.
噬菌体抗体技术可用于产生针对几乎任何抗原的人源抗体。许多治疗性靶抗原是细胞表面受体，这对于抗体的产生来说可能是具有挑战性的靶点。此外，对于许多治疗应用而言，人们需要的抗体不仅能结合细胞表面受体，而且在结合后能被内化进入细胞。这使得可以利用抗体将一系列有效载荷递送至细胞内以实现治疗效果。在本章中，我们描述了如何直接在肿瘤细胞系上筛选人源噬菌体抗体文库，以产生能结合细胞表面受体且在结合后迅速被内化进入细胞的抗体。具体方案展示了如何：（1）直接从人源噬菌体抗体文库中筛选细胞结合和内化抗体；（2）在高通量流式细胞术检测中筛选噬菌体抗体与用于筛选的肿瘤细胞系的结合情况；（3）使用免疫沉淀和质谱鉴定噬菌体抗体所结合的抗原；（4）通过先在肿瘤细胞系上进行连续筛选，然后在酵母表面展示靶肿瘤抗原的酵母上进行筛选，将细胞结合和内化筛选导向特定的肿瘤抗原。
