In vitro antibody discovery and/or affinity maturation are often performed using antibody fragments (Fabs), but most monovalent Fabs are reformatted as bivalent IgGs (monoclonal antibodies, mAbs) for therapeutic applications. One problem related to reformatting antibodies is that the bivalency of mAbs can lead to increased antibody self-association and poor biophysical properties (e.g., reduced antibody solubility and increased viscosity). Therefore, it is important to identify monovalent Fabs early in the discovery and/or optimization process that will display favorable biophysical properties when reformatted as bivalent mAbs. Here we demonstrate a facile approach for evaluating Fab self-association in a multivalent assay format that is capable of identifying antibodies with low self-association and favorable colloidal properties when reformatted as bivalent mAbs. Our approach (self-interaction nanoparticle spectroscopy, SINS) involves immobilizing Fabs on gold nanoparticles in a multivalent format (multiple Fabs per nanoparticle) and evaluating their self-association behavior via shifts in the plasmon wavelength or changes in the absorbance values. Importantly, we find that SINS measurements of Fab self-association are correlated with self-interaction measurements of bivalent mAbs and are useful for identifying antibodies with favorable biophysical properties. Moreover, the significant differences in the levels of self-association detected for Fabs and mAbs with similar frameworks can be largely explained by the physicochemical properties of the complementarity-determining regions (CDRs). Comparison of the properties of the CDRs in this study relative to those of approved therapeutic antibodies reveals several key factors (net charge, fraction of charged residues, and presence of self-interaction motifs) that strongly influence antibody self-association behavior. Increased positive charge in the CDRs was observed to correlate with increased risk of high self-association for the mAbs in this study and clinical-stage antibodies. We expect that these findings will be useful for improving the development of therapeutic antibodies that are well suited for high concentration applications.

中文翻译：

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基于生物物理和基于序列的方法来鉴定具有高胶体稳定性的单价和二价抗体

体外抗体发现和/或亲和力成熟通常使用抗体片段（Fab）进行，但大多数单价Fab被重新格式化为二价IgG（单克隆抗体，mAb）以用于治疗应用。与抗体重新格式化有关的一个问题是，mAb的双价性可能导致抗体自缔合增加和生物物理特性变差（例如，抗体溶解度降低和粘度增加）。因此，重要的是在发现和/或优化过程中尽早鉴定单价Fab，当将其重新格式化为二价mAb时将显示出有利的生物物理特性。在这里，我们展示了一种以多价测定形式评估Fab自缔合的简便方法，当重新格式化为二价mAb时能够鉴定具有低自缔合和有利胶体性质的抗体。我们的方法（自相互作用纳米粒子光谱法，SINS）涉及以多价形式（每个纳米粒子多个Fab）将Fabs固定在金纳米颗粒上，并通过等离激元波长的改变或吸光度值的变化评估其自缔合行为。重要的是，我们发现Fab自缔合的SINS测量与二价mAb的自相互作用测量相关，可用于鉴定具有良好生物物理特性的抗体。而且，具有相似框架的Fab和mAb的自缔合水平检测到的显着差异可以通过互补决定区（CDR）的物理化学性质来很大程度上解释。相对于已批准的治疗性抗体，本研究中CDR的特性进行比较，发现了几个关键因素（净电荷，带电残基的分数和自相互作用基序的存在）会严重影响抗体的自缔合行为。在此研究中，观察到CDR中正电荷的增加与mAb高度自缔合的风险增加以及临床阶段抗体的增加有关。我们希望这些发现将有助于改善非常适合高浓度应用的治疗性抗体的开发。