For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 μg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

中文翻译：

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增强糖工程水稻细胞生产的曲妥珠单抗的疗效


几十年来，人们一直提出将植物基表达系统作为生产生物制药（包括治疗性单克隆抗体 （mAb））的替代平台，但与植物基生物制药中附着的植物特异性 N-糖相关的免疫原性问题尚未完全解决。为了消除所有植物特异性 N-糖结构，使用 CRISPR/Cas9 系统在水稻 （Oryza sativa） 中突变了 8 个参与植物特异性 N-糖基化的基因。糖工程细胞系 PhytoRice® 含有主要的 GnGn （G0） 糖型。然后通过农杆菌共培养将密码子优化的曲妥珠单抗 （TMab） 基因引入 PhytoRice®。对选定的细胞系进行悬浮培养，并从培养的培养基中纯化细胞分泌的 TMab。PhytoRice® （P-TMab） 产生的 TMab 的氨基酸序列与 TMab 相同。P-TMab 对 BT-474 癌细胞系增殖的抑制作用在浓度高于 1 μg/mL 时显著增强 （****P < 0.0001）。P-TMab 与 FcγRIIIa 变体 FcγRIIIa-F158 结合的效率是 TMab 的 2.7 倍以上。在体外 ADCC 测定中，P-TMab 对 Jurkat 细胞的 ADCC 疗效比 TMab 高 2.6 倍。此外，在使用 BT-474 小鼠模型的异种移植试验中，与 TMab 相比，P-TMab 显示出有效的肿瘤摄取和较少的肝脏摄取。这些结果表明，与当前的 CHO 细胞相比，糖工程 PhytoRice® 可以成为 mAb 生产的替代平台，并且与 TMab 相比，P-TMab 具有新颖且增强的功效。