Müller Dafne, Karle Anette, Meissburger Bettina, Höfig Ines, Stork Roland, Kontermann Roland E
Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
J Biol Chem. 2007 Apr 27;282(17):12650-60. doi: 10.1074/jbc.M700820200. Epub 2007 Mar 8.
Recombinant bispecific antibodies such as tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb) have shown to be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, therapeutic efficacy is hampered by a short serum half-life of these small molecules having molecule masses of 50-60 kDa. Thus, improvement of the pharmacokinetic properties of small bispecific antibody formats is required to enhance efficacy in vivo. In this study, we generated several recombinant bispecific antibody-albumin fusion proteins and analyzed these molecules for biological activity and pharmacokinetic properties. Three recombinant antibody formats were produced by fusing two different scFv molecules, bispecific scDb or taFv molecules, respectively, to human serum albumin (HSA). These constructs (scFv(2)-HSA, scDb-HSA, taFv-HSA), directed against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor complex molecule CD3, retained full binding capacity to both antigens compared with unfused scFv, scDb, and taFv molecules. Tumor antigen-specific retargeting and activation of T cells as monitored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv(2)-HSA. T cell activation could be further enhanced by a target cell-specific costimulatory signal provided by a B7-DbCEA fusion protein. Furthermore, we could demonstrate that fusion to serum albumin strongly increases circulation time of recombinant bispecific antibodies. In addition, our comparative study indicates that single chain diabody-albumin fusion proteins seem to be the most promising format for further studying cytotoxic activities in vitro and in vivo.
重组双特异性抗体，如串联单链抗体片段（taFv）、双体（Db）或单链双体（scDb），已显示出能够将T淋巴细胞重新靶向肿瘤细胞，从而导致肿瘤细胞的破坏。然而，这些分子量为50 - 60 kDa的小分子在血清中的半衰期较短，限制了其治疗效果。因此，需要改善小型双特异性抗体形式的药代动力学特性，以提高其体内疗效。在本研究中，我们制备了几种重组双特异性抗体 - 白蛋白融合蛋白，并分析了这些分子的生物活性和药代动力学特性。通过将两种不同的单链抗体片段、双特异性单链双体或串联单链抗体片段分子分别与人血清白蛋白（HSA）融合，制备了三种重组抗体形式。这些构建体（scFv(2)-HSA、scDb-HSA、taFv-HSA）针对肿瘤抗原癌胚抗原（CEA）和T细胞受体复合物分子CD3，与未融合的单链抗体片段、双体和串联单链抗体片段分子相比，保留了对两种抗原的完全结合能力。通过白细胞介素 - 2释放监测发现，scDb、scDb-HSA、taFv-HSA以及程度较轻的scFv(2)-HSA均能实现肿瘤抗原特异性的T细胞重新靶向和激活。B7-DbCEA融合蛋白提供的靶细胞特异性共刺激信号可进一步增强T细胞的激活。此外，我们还证明，与血清白蛋白融合可显著延长重组双特异性抗体的循环时间。此外，我们的比较研究表明，单链双体 - 白蛋白融合蛋白似乎是进一步研究体外和体内细胞毒性活性最有前景的形式。
