BACKGROUND Bispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing. METHODS We have designed a solution in which the CH1 and CL domain pair in one of the Fab fragments is replaced with a CH3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII). RESULTS Bispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains. CONCLUSION Incorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties. GENERAL SIGNIFICANCE Our results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with CH3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing.

中文翻译：

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恒定结构域交换 Fab 使异二聚体双特异性 SEED 抗体中的特定轻链配对成为可能。

背景技术双特异性抗体有望广泛扩展单克隆抗体技术的临床应用。已经建立了几种用于重链异二聚化的方法来生产具有两个不同 Fab 臂的抗体，但重链和轻链的混杂配对仍然是其制造的挑战。方法 我们设计了一种解决方案，其中一个 Fab 片段中的 CH1 和 CL 结构域对被替换为 CH3 结构域对并进行异二聚化，以促进基于链交换工程结构域的双特异性异二聚体抗体中正确修饰的 Fab 链配对(SEED) 支架，对上皮生长因子受体和 CD3 或 CD16 (FcγRIII) 具有特异性。结果双特异性抗体保留与其靶抗原的结合并重定向原代T细胞或NK细胞以诱导对靶细胞的有效杀伤。所有抗体在 Expi293F 细胞中均以高产量表达，在尺寸排阻色谱中检测为单个尖锐对称峰并保持高热稳定性。质谱分析揭示了双特异性 SEED 抗体以及与两条不同竞争轻链共表达的单臂 SEED 抗体的特定重链到轻链配对。结论 将恒定结构域交换的 Fab 片段掺入 SEED 抗体会产生具有良好生物物理特性的功能分子。