Lu Dan, Jimenez Xenia, Zhang Haifan, Atkins Amanda, Brennan Laura, Balderes Paul, Bohlen Peter, Witte Larry, Zhu Zhenping
Department of Antibody Technology, ImClone Systems Incorporated, New York, NY 10014, USA.
J Immunol Methods. 2003 Aug;279(1-2):219-32. doi: 10.1016/s0022-1759(03)00251-5.
The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens. Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH(3) domain of an IgG. The novel BsAb, which we termed "di-diabody", represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH(3) domains. A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography. Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors. The di-diabody retained good antigen-binding activity after incubation at 37 degrees C in mouse serum for 72 h, demonstrating good product stability. Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity. This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.
双特异性抗体（BsAb）作为治疗药物的临床开发一直受到传统方法难以制备足够数量和质量材料的阻碍。近年来，已经开发了多种重组方法来高效生产BsAb，包括抗体片段和全长IgG样分子。这些重组抗体分子具有双抗原结合能力，在大多数情况下，对其每个靶抗原具有单价性。在这里，我们描述了一种通过将双特异性/二价双抗体通过铰链区与IgG的CH(3)结构域的N端进行基因融合来生产新型四价BsAb的有效方法，该BsAb对其每个靶抗原都有两个抗原结合位点。我们将这种新型BsAb称为“双二体”，它代表了一种由铰链区和CH(3)结构域之间二聚化产生的四价双体二聚体。使用针对血管内皮生长因子的两种酪氨酸激酶受体的两种抗体构建了双二体，该双二体在单个大肠杆菌宿主和哺乳动物细胞中均有表达，并通过一步亲和层析纯化至同质。与双特异性/二价双抗体相比，四价双二体对其两种靶抗原的结合更有效，并且在阻断配体与受体结合方面更有效。双二体在小鼠血清中于37℃孵育72小时后仍保留良好的抗原结合活性，表明其具有良好的产品稳定性。最后，双二体在哺乳动物细胞中的表达产生了更高的产量和更好的抗体活性。这种BsAb片段的设计和表达应该适用于任何一对抗原特异性。
