Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity.

由于近年来产生双特异性抗体（BsAb）的技术创新，BsAb已成为诊断和治疗应用的重要试剂。然而，难以生产由两个不同的臂组成的具有高产率和纯度的异二聚体构成了它们在学术和临床应用中的主要限制。在这里，我们描述了一种由常规抗原结合片段（Fab）和单域抗体（sdAb）组成的新颖的含Fc的BsAb格式（Fab×sdAb-Fc），可避免抗体过程中的轻链错配部件。在这项研究中，Fab x sdAb-Fc BsAb通过三种广泛使用的重链异二聚方法有效生产：旋钮插入孔（KIH），电荷对（CP）和受控Fab臂交换（cFAE）分别。新颖的Fab x sdAb-Fc形式提供了一种快速高效的策略来生成具有高纯度的BsAb，并提供了一种基于分子量（MW）从不需要的抗体中进一步纯化所需BsAb的独特可能性。与传统的BsAb形式相比，Fab x sdAb-Fc形式的优势可能因此提供了直接的机会，可以将双特异性抗体原理应用于疾病和疾病（例如癌症和自身免疫性）的新型靶标和途径的研究和开发。