Benedetti Filippo, Stracke Florian, Stadlmayr Gerhard, Stadlbauer Katharina, Rüker Florian, Wozniak-Knopp Gordana
CD Laboratory for Innovative Immunotherapeutics, Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190, Vienna, Austria.
Biochem Biophys Rep. 2021 Feb 27;26:100959. doi: 10.1016/j.bbrep.2021.100959. eCollection 2021 Jul.
Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb format where a set of mutated amino acid residues in the C3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab C1/C domain pair with a pair of antigen-binding C3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding C3 domains. Such bispecific molecules were produced in a "Fab-like" format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding C3 domains offer an alternative solution in positioning and valency of antigen binding sites.
单克隆抗体可通过融合方法、修饰其互补决定区（CDR）环，也可通过修饰其恒定区来获得与第二种抗原结合的特性，例如在单克隆抗体形式中，C3结构域中的一组突变氨基酸残基能够与第二种抗原进行高亲和力特异性相互作用。我们测试了在具有曲妥珠单抗可变区和血管内皮生长因子（VEGF）结合C3结构域的模型支架中，用一对抗原结合C3结构域取代Fab C1/C结构域对来引入第二种抗原多个结合位点的可能性。此类双特异性分子以“类Fab”形式和全长抗体形式产生。使用质谱分析，新型构建体具有预期的分子组成。它们在标准实验室条件下高水平表达，通过蛋白A和凝胶过滤作为单体纯化，并且具有高热稳定性。它们保留了对两种靶抗原的高亲和力结合。最后，由于与内源性分泌细胞因子的交联水平提高，Her2/VEGF结合结构域交换的双特异性抗体能够介导对Her2过表达细胞系SK-BR-3增强的表面Her2内化作用。总之，具有交换抗原结合C3结构域的Fab的双特异性抗体在抗原结合位点的定位和价态方面提供了一种替代解决方案。
