Heitner Tara, Satozawa Noboru, McLean Kirk, Vogel David, Cobb Ronald R, Liu Bing, Mahmoudi Mithra, Finster Silke, Larsen Brent, Zhu Ying, Zhou Hongxing, Müller-Tiemann Beate, Monteclaro Felipe, Zhao Xiao-Yan, Light David R
Berlex Biosciences, Richmond, California 94806, USA.
J Biomol Screen. 2006 Dec;11(8):985-95. doi: 10.1177/1087057106293841. Epub 2006 Nov 7.
A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.
利用多价噬菌体展示技术分离得到的一种治疗性抗体候选物（AT-19）以多聚体而非单体形式天然结合调节素（TR）。本报告强调了在天然抗原上筛选和选择噬菌体抗体的重要性，并再次强调了使用单体噬菌体展示系统进行筛选时可能会丢弃潜在有价值抗体的可能性。本文描述了一种详细的活细胞淘选选择和筛选方法，用于分离多价活性抗体。AT-19是一种完全人源抗体，可识别细胞表面蛋白TR，TR是治疗性抗体内化的一个潜在前列腺癌靶点。AT-19是从用超噬菌体拯救的多价单链可变片段（scFv）抗体库中分离得到的。荧光激活细胞分选数据支持分离AT-19所需的多价性，该数据表明多价AT-19噬菌体颗粒在高噬菌体浓度下具有结合能力，而单价颗粒则无法结合。纯单体scFv AT-19不与细胞上的天然受体结合，而二聚体scFv或免疫球蛋白G则以纳摩尔亲和力结合。AT-19抗体对天然TR具有专一性二价结合活性，这归因于噬菌体上scFv的多价展示以及通过交替使用两种重组细胞系进行选择和筛选的方法。
