当过表达的抗体具有固有的高缩合倾向时,明显的包涵体可以在内质网(ER)中发展。这些观察结果表明,被认为显示出显著溶解度问题的抗体可以在抗体过表达期间以ER相关包涵体的形式预先揭示此类特征。为了定义溶解度问题和包涵体表型之间的关系,我们研究了一个模型人IgG2λ的生物合成,该模型在酸性制剂缓冲液中显示出严重的乳光,但在生理pH下仍保持高溶解度。与pH依赖性溶解度特性一致,模型抗体在ER腔的生理pH环境中没有诱导明显的包涵体。然而,当抗体的各个亚基链分别表达时,轻链(LC)在ER中自发诱导明显的晶体状包涵体。通过在生理pH下简单地浓缩纯化的LC蛋白,LC结晶事件在体外是容易再现的。通过监测氨基酸取代对细胞内LC晶体形成的影响,通过合理的诱变方法鉴定了LC结晶的两个独立结构决定因素。还使用纯化的LC蛋白在体外概括了突变对结晶的影响。重要的是,当直接引入模型抗体时,阻止LC结晶的突变修复了抗体的溶解度问题,而不损害分泌输出或抗原结合。这些结果表明,ER可以作为“生理试管”,不仅报告分泌货物在生理pH值下的高冷凝倾向,而且还提供了指导抗体工程策略的正交方法。
Prominent inclusion bodies can develop in the endoplasmic reticulum (ER) when overexpressed antibodies possess intrinsically high condensation propensities. These observations suggest that antibodies deemed to show notable solubility problems may reveal such characteristics preemptively in the form of ER-associated inclusion bodies during antibody overexpression. To define the relationships between solubility problems and inclusion body phenotypes, we investigated the biosynthesis of a model human IgG2λ that shows severe opalescence in an acidic formulation buffer yet retains high solubility at physiological pH. Consistent with the pH-dependent solubility characteristics, the model antibody did not induce notable inclusion body in the physiological pH environment of the ER lumen. However, when individual subunit chains of the antibody were expressed separately, the light chain (LC) spontaneously induced notable crystal-like inclusion bodies in the ER. The LC crystallization event was readily reproducible in vitro by simply concentrating the purified LC protein at physiological pH. Two independent structural determinants for the LC crystallization were identified through rational mutagenesis approach by monitoring the effect of amino acid substitutions on intracellular LC crystallogenesis. The effect of mutations on crystallization was also recapitulated in vitro using purified LC proteins. Importantly, when introduced directly into the model antibody, a mutation that prevents the LC crystallization remediated the antibody\'s solubility problem without compromising the secretory output or antigen binding. These results illustrate that the ER can serve as a \"physiological test tube\" that not only reports secretory cargo\'s high condensation propensity at physiological pH, but also provides an orthogonal method that guides antibody engineering strategy.