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.

当过度表达的抗体具有固有的高缩合倾向时，可以在内质网 (ER) 中形成突出的包涵体。这些观察结果表明，被认为表现出显着溶解度问题的抗体可能会在抗体过表达期间以 ER 相关包涵体的形式先发制人地揭示这些特征。为了确定溶解度问题与包涵体表型之间的关系，我们研究了模型人 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白色，但在生理 pH 值下仍保持高溶解度。与 pH 依赖性溶解度特性一致，模型抗体在内质网腔的生理 pH 环境中不诱导显着的包涵体。然而，当抗体的各个亚基链单独表达时，轻链 (LC) 会自发地在 ER 中诱导显着的晶体状包涵体。通过在生理 pH 值下简单地浓缩纯化的 LC 蛋白，LC 结晶事件很容易在体外重现。通过监测氨基酸取代对细胞内 LC 结晶的影响，通过合理的诱变方法确定了 LC 结晶的两个独立结构决定因素。还使用纯化的 LC 蛋白在体外重现了突变对结晶的影响。重要的是，当直接引入模型抗体时，防止 LC 结晶的突变修复了抗体的溶解性问题，而不会影响分泌输出或抗原结合。