Nagata Satoshi, Salvatore Giuliana, Pastan Ira
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr, Rm 5106, Bethesda, MD 20892-4264, USA.
J Immunol Methods. 2003 Sep;280(1-2):59-72. doi: 10.1016/s0022-1759(03)00192-3.
Recent advancements in antibody-based therapies require the development of an efficient method for generation of monoclonal antibodies (MAbs) against the native form of membrane proteins. We examined DNA immunization followed by a single boost with cells as a protein-free immunization protocol for production of MAbs. Mice immunized with plasmid cDNAs encoding human CD30 or Ret tyrosine kinase were given a single boost with cells expressing the corresponding antigen prior to cell fusion. A total of nine cell fusion experiments revealed that the cell boost is necessary for efficient generation of hybridomas and the DNA-cell boost method gave good yields of specific MAbs (5-59 MAbs from one mouse). All IgG isotypes except IgG3 were generated, although IgG2a was the dominant isotype. All the MAbs reacted with native antigens expressed on cells in a fluorescence-activated cell sorter (FACS) analysis as well as with recombinant CD30 or Ret protein genetically fused with human Fc in an enzyme-linked immunosorbent assay (ELISA). The affinities of the anti-CD30 MAbs to CD30-Fc protein ranged from 0.9 to 12.4 nM Kds, which were comparable to existing MAbs to these proteins, which range from 3.0 to 13.0 nM. Western blot analysis and topographical epitope mapping experiments based on the mutual competition of pairs of the anti-CD30 MAbs revealed that about 40% of the epitopes were linear epitopes and that each epitope was topographically classified into one of six groups. The large number of MAbs that react with high affinities to a variety of epitopes on the native form of antigens indicates that the method presented in this paper could be generally useful for generating MAbs to other membrane proteins.
基于抗体的治疗方法的最新进展要求开发一种高效的方法来生成针对膜蛋白天然形式的单克隆抗体（MAb）。我们研究了DNA免疫，随后用细胞进行单次加强免疫，作为一种无蛋白免疫方案来生产MAb。在用编码人CD30或Ret酪氨酸激酶的质粒cDNA免疫的小鼠中，在细胞融合前用表达相应抗原的细胞进行单次加强免疫。总共九次细胞融合实验表明，细胞加强免疫对于高效产生杂交瘤是必要的，并且DNA-细胞加强免疫方法产生了良好产量的特异性MAb（每只小鼠产生5-59个MAb）。除IgG3外的所有IgG同种型均有产生，尽管IgG2a是主要的同种型。在荧光激活细胞分选仪（FACS）分析中，所有MAb均与细胞上表达的天然抗原反应，并且在酶联免疫吸附测定（ELISA）中与与人Fc基因融合的重组CD30或Ret蛋白反应。抗CD30 MAb与CD30-Fc蛋白的亲和力范围为0.9至12.4 nM Kds，与针对这些蛋白的现有MAb相当，后者范围为3.0至13.0 nM。基于抗CD30 MAb对之间的相互竞争的蛋白质印迹分析和拓扑表位作图实验表明，约40％的表位是线性表位，并且每个表位在拓扑上分为六个组之一。大量与抗原天然形式上的多种表位具有高亲和力反应的MAb表明，本文提出的方法通常可用于生成针对其他膜蛋白的MAb。
