Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
Plant Biotechnol J. 2014 Dec;12(9):1333-42. doi: 10.1111/pbi.12251. Epub 2014 Sep 4.
The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-α, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1κ-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ- and IgG1κ-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1κ-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, β1,2-xylose and core α1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core α1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.
IgA 的独特特征，如招募中性粒细胞和抑制 IgG 和 IgE 介导的炎症反应的能力，使其成为几种治疗应用有前途的抗体类型。然而，与 IgG 相比，关于植物生产 IgA 的报道很少。我们在本氏烟中生产了三种针对促炎细胞因子的治疗性抗体的 IgA1κ 和 IgG1κ 版本：针对 TNF-α 的英夫利昔单抗和阿达木单抗，以及针对白细胞介素-12p40 亚基的乌司奴单抗。我们评估了抗体的产量、质量和 N-糖基化。所有六种抗体的表达水平在总可溶性蛋白含量的 3.5%至 9%之间相当，并且在细胞测定中显示出中和活性。然而，与 IgG 相比，基于 IgA1κ 的阿达木单抗和乌司奴单抗的分泌能力较差。无论抗体类型如何，英夫利昔单抗的分泌能力都较差。这与观察到的 IgA1κ 和 IgG1κ 基于英夫利昔单抗富含寡甘露糖型 N-聚糖结构的结果一致。对于 IgG1κ 基于的乌司奴单抗和阿达木单抗，主要的 N-聚糖类型是典型的植物复杂 N-聚糖，双天线，末端 N-乙酰葡萄糖胺，β1,2-木糖和核心α1,3-岩藻糖。相比之下，基于 IgA 的抗体的主要 N-聚糖是木糖基化的，但缺乏核心α1,3-岩藻糖和一个末端 N-乙酰葡萄糖胺。这种类型的 N-聚糖在植物中通常以边缘百分比出现，从未被证明是植物生产的重组蛋白的主要部分。我们的数据表明，抗体类型可能对抗体接受的 N-聚糖类型产生深远影响。
