Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever.
Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever.
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DOI：
10.3389/fpls.2022.925008
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发表时间：
2022
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
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单克隆抗体 (mAb) 是从诊断到治疗的许多生命科学应用中使用的重要蛋白质。不同应用对单克隆抗体的高需求促使开发快速、可靠的重组生产平台。植物提供了一种快速且廉价的重组单克隆抗体生产系统。此外，当与已建立的单克隆抗体发现平台配合使用时，可以轻松定制植物以生产针对同一靶标的不同同种型的单克隆抗体。在这里，我们证明杂交瘤产生的针对几丁质酶 1 (CTS1)（球孢子菌属抗原）的小鼠单克隆抗体可以通过生物工程设计，与使用植物表达的球孢子菌病（谷热）血清学诊断试剂盒一起使用。最初的小鼠 IgG 在糖工程烟草植物中进行修饰和重组，通过瞬时表达为具有人 kappa、gamma 和 mu 恒定区的 IgG 和 IgM 同种型。使用与食品和药物管理局 (FDA) 批准的谷热诊断试剂盒类似的试剂，植物中产生的两种 mAb 同种型能够维持对 CTS1 的靶抗原识别。由于目前批准的试剂盒均不提供抗体稀释对照，因此与诊断抗原制剂的主要成分 CTS1 结合的抗体人源化可能为谷热诊断缺乏一致反应性抗体对照提供解决方案。此外，我们的工作为重组单克隆抗体的可重复和一致生产奠定了基础，该重组单克隆抗体被设计为具有用于诊断测定的特定同种型。
Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in glycoengineered Nicotiana benthamiana plants via transient expression as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.