单克隆抗体可以通过融合方法或其CDR环的修饰获得第二抗原的接合特性。而且还通过修改它们的恒定域,例如在mAb2形式中,其中CH3结构域中的一组突变的氨基酸残基实现与第二抗原的高亲和力特异性相互作用。我们测试了通过在具有曲妥珠单抗可变结构域和VEGF结合CH3结构域的模型支架中用一对抗原结合CH3结构域替换FabCH1/CL结构域对引入第二抗原的多个结合位点的可能性。此类双特异性分子以“Fab样”形式和全长抗体形式产生。使用质谱法,新的构建体具有预期的分子组成。它们在标准实验室条件下表现得很高,用蛋白A和凝胶过滤纯化为单体,具有高热稳定性。保留了它们与两种靶抗原的高亲和力结合。最后,由于与内源性分泌的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过表达的细胞系SK-BR-3增强的表面Her2内化作用。最后,具有以交换的抗原结合CH3结构域为特征的Fab的双特异性抗体在抗原结合位点的定位和效价方面提供了替代解决方案。
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 mAb2 format where a set of mutated amino acid residues in the CH3 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 CH1/CL domain pair with a pair of antigen-binding CH3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding CH3 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 CH3 domains offer an alternative solution in positioning and valency of antigen binding sites.