Human cytomegalovirus-specific T-cell receptor engineered for high affinity and soluble expression using mammalian cell display.
Human cytomegalovirus-specific T-cell receptor engineered for high affinity and soluble expression using mammalian cell display.
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人巨细胞病毒特异性 T 细胞受体经过工程设计，可利用哺乳动物细胞展示实现高亲和力和可溶性表达。
DOI：
10.1074/jbc.ra118.007187
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Maynard,JenniferA
中科院分区：
文献类型：
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作者：
Wagner,EllenK;Qerqez,AhlamN;Stevens,ChristopherA;Nguyen,AnnaleeW;Delidakis,George;Maynard,JenniferA
T 细胞受体 (TCR) 作为治疗剂和抗体样试剂在监测疾病进展和疫苗功效方面具有巨大潜力。抗体只能识别分泌的和表面结合的蛋白质，而 TCR 则可以识别以其他方式无法接近的疾病相关细胞内蛋白质，当它们以与主要组织相容性复合体 (pMHC) 结合的加工肽形式呈现时。 TCR 主要用于癌症治疗应用，但也可以针对巨细胞病毒 (CMV) 等传染病。然而，TCR 比抗体更难表达和设计，需要先进的方法来使其广泛使用。在这里，我们设计了人类 CMV 特异性 TCR RA14，以实现高亲和力和稳健的可溶性表达。为了实现这一目标，我们调整了之前报道的哺乳动物展示系统来呈现 TCR 胞外结构域，并使用它来筛选 CDR3 文库，以寻找具有增加的 pMHC 亲和力的克隆。经过三轮选择后，特征克隆在人 Jurkat T 细胞表面表达时保留了肽特异性和激活性。我们通过将 TCR 胞外结构域与抗体铰链和 Fc 恒定区融合，在恒定结构域之间添加稳定的二硫键并破坏预测的糖基化位点，获得了高产量的可溶性单体蛋白。通过表面等离振子共振测量，一种变体对其同源 pMHC 表现出 50 nm 的亲和力，并且对呈递该 pMHC 的细胞进行了特异性染色。我们的工作已经确定了一种对免疫显性 CMV 肽具有高亲和力的人类 TCR，并提供了一种快速设计用于生物医学应用的可溶性 TCR 的新策略。
T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV). However, TCRs are more difficult to express and engineer than antibodies, and advanced methods are needed to enable their widespread use. Here, we engineered the human CMV–specific TCR RA14 for high-affinity and robust soluble expression. To achieve this, we adapted our previously reported mammalian display system to present TCR extracellular domains and used this to screen CDR3 libraries for clones with increased pMHC affinity. After three rounds of selection, characterized clones retained peptide specificity and activation when expressed on the surface of human Jurkat T cells. We obtained high yields of soluble, monomeric protein by fusing the TCR extracellular domains to antibody hinge and Fc constant regions, adding a stabilizing disulfide bond between the constant domains and disrupting predicted glycosylation sites. One variant exhibited 50 nmaffinity for its cognate pMHC, as measured by surface plasmon resonance, and specifically stained cells presenting this pMHC. Our work has identified a human TCR with high affinity for the immunodominant CMV peptide and offers a new strategy to rapidly engineer soluble TCRs for biomedical applications.