Bruynck A, Seemann G, Bosslet K
Research Laboratories of Behringwerke AG, Marburg/Lahn, Germany.
Br J Cancer. 1993 Mar;67(3):436-40. doi: 10.1038/bjc.1993.84.
A humanised bispecific monoclonal antibody (bsMAb) with binding specificity for carcinoembryonic antigen (CEA) on one arm and a radiolabelled chelate (DTPA-90Y) on the other arm was generated by consecutively transfecting the humanised genes of an anti-CEA MAb and the chimerised genes of an anti-chelate MAb into eucaryotic BHK cells using the calcium-phosphate coprecipitation technique. The antibodies secreted were of IgG3 isotype with a shortened hinge region (delta gamma 3) and light chains. Double transfectomas were screened for the secretion of bsMAbs using a double determinant enzyme-linked immunosorbent assay (ELISA) based on solid phase attached HSA-benzyl-DTPA and an anti-idiotypic MAb selective for the CEA-specific arm. After purification on two immunoaffinity chromatography columns, the humanised bsMAbs were characterised by SDS-PAGE and a quantitative binding assay in antigen excess. The purification procedure resulted in 95% reactive bispecific MAb. This humanised bsMAb may be employed in two phase radioimmunotherapy, binding to the tumour via the anti-CEA arm and localising a radiolabelled chelate with the other arm, without inducing a strong immune response observed sometimes with murine MAbs.
通过使用磷酸钙共沉淀技术，将抗癌胚抗原（CEA）单克隆抗体（MAb）的人源化基因和抗螯合剂MAb的嵌合基因依次转染到真核BHK细胞中，产生了一种人源化双特异性单克隆抗体（bsMAb），其一条臂对癌胚抗原（CEA）具有结合特异性，另一条臂带有放射性标记的螯合物（DTPA-90Y）。分泌的抗体为IgG3同种型，具有缩短的铰链区（δγ3）和轻链。使用基于固相连接的HSA-苄基-DTPA和对CEA特异性臂具有选择性的抗独特型MAb的双决定簇酶联免疫吸附测定（ELISA）筛选双转染瘤分泌的bsMAb。在两根免疫亲和色谱柱上纯化后，通过SDS-PAGE和抗原过量的定量结合测定对人源化bsMAb进行表征。纯化过程产生了95%具有反应活性的双特异性MAb。这种人源化bsMAb可用于两阶段放射免疫治疗，通过抗CEA臂与肿瘤结合，并用另一臂定位放射性标记的螯合物，而不会像鼠源MAb有时那样引发强烈的免疫反应。
