Robust production of monovalent bispecific IgG antibodies through novel electrostatic steering mutations at the C(H)1-C(λ) interface.

Bispecific antibodies represent an increasingly large fraction of biologics in therapeutic development due to their expanded scope in functional capabilities. Asymmetric monovalent bispecific IgGs (bsIgGs) have the additional advantage of maintaining a native antibody-like structure, which can provide favorable pharmacology and pharmacokinetic profiles. The production of correctly assembled asymmetric monovalent bsIgGs, however, is a complex engineering endeavor due to the propensity for non-cognate heavy and light chains to mis-pair. Previously, we introduced the DuetMab platform as a general solution for the production of bsIgGs, which utilizes an engineered interchain disulfide bond in one of the CH1-CL domains to promote orthogonal chain pairing between heavy and light chains. While highly effective in promoting cognate heavy and light chain pairing, residual chain mispairing could be detected for specific combinations of Fv pairs. Here, we present enhancements to the DuetMab design that improve chain pairing and production through the introduction of novel electrostatic steering mutations at the CH1-CL interface with lambda light chains (CH1-Cλ). These mutations work together with previously established charge-pair mutations at the CH1-CL interface with kappa light chains (CH1-Cκ) and Fab disulfide engineering to promote cognate heavy and light chain pairing and enable the reliable production of bsIgGs. Importantly, these enhanced DuetMabs do not require engineering of the variable domains and are robust when applied to a panel of bsIgGs with diverse Fv sequences. We present a comprehensive biochemical, biophysical, and functional characterization of the resulting DuetMabs to demonstrate compatibility with industrial production benchmarks. Overall, this enhanced DuetMab platform substantially streamlines process development of these disruptive biotherapeutics.

双特异性抗体在治疗性开发的生物制品中所占比例日益增大，这是因为它们在功能能力方面的应用范围有所扩大。不对称单价双特异性IgG（bsIgG）具有保持天然抗体样结构的额外优势，这能够提供良好的药理学和药代动力学特性。然而，由于非同源重链和轻链容易错配，正确组装不对称单价bsIgG的生产是一项复杂的工程难题。此前，我们引入了DuetMab平台作为生产bsIgG的通用解决方案，该平台利用其中一个CH1 - CL结构域中的工程化链间二硫键来促进重链和轻链之间的正交链配对。虽然在促进同源重链和轻链配对方面非常有效，但对于特定的Fv对组合仍可检测到残留的链错配。在此，我们介绍了对DuetMab设计的改进，通过在与λ轻链（CH1 - Cλ）的CH1 - CL界面引入新的静电导向突变来改善链配对和生产。这些突变与先前在与κ轻链（CH1 - Cκ）的CH1 - CL界面建立的电荷对突变以及Fab二硫键工程共同作用，以促进同源重链和轻链配对，并使bsIgG能够可靠地生产。重要的是，这些增强的DuetMab不需要对可变结构域进行工程改造，并且在应用于具有不同Fv序列的一组bsIgG时具有稳健性。我们对所得的DuetMab进行了全面的生化、生物物理和功能特性分析，以证明其与工业生产标准相符。总体而言，这个增强的DuetMab平台极大地简化了这些突破性生物治疗药物的工艺开发。