Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core α1,3-fucose and β1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFc^ΔXF, has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumour mouse model, we found that systemic administration of AvFc^ΔXF, but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumours, suggesting that Fc-mediated effector functions were integral. AvFc^ΔXF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of antidrug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFc^ΔXF before implanting tumours. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFc^ΔXF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFc's antitumor activity.

中文翻译：

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糖工程和抗药抗体对植物凝集素-Fc融合蛋白抗癌活性的影响

植物是制造糖工程单克隆抗体和抗体样分子的高效生产平台。Avaren-Fc (AvFc) 是一种在本氏烟草中产生的凝集素-Fc 融合蛋白或凝集体，可选择性识别癌症相关的高甘露糖聚糖。在这项研究中，我们报告了 AvFc 糖变体的产生，该糖变体不含植物聚糖，包括核心 α1,3-岩藻糖和 β1,2-木糖残基。通过使用 HPLC 的聚糖分析证实了这些聚糖的成功去除。这种变体，AvFc ^ΔXF，对 Fc γ 受体具有显着更高的亲和力，并在针对 B16F10 鼠黑色素瘤细胞的抗体依赖性细胞介导的细胞毒性 (ADCC) 报告基因测定中诱导更高水平的荧光素酶表达，而不会诱导细胞凋亡或抑制增殖。在 B16F10 侧腹肿瘤小鼠模型中，我们发现 AvFc ^ΔXF的全身给药，但不是对 Fc 受体缺乏亲和力的非糖基化 AvFc 变体，显着延迟了肿瘤的生长，表明 Fc 介导的效应功能是不可或缺的。AvFc ^ΔXF治疗还显着减少了 B16F10 在静脉内攻击时的肺转移，而糖结合缺陷型突变体未能显示出疗效。最后，我们确定了抗药物抗体 (ADA) 对药物活性的影响通过在植入肿瘤之前用 AvFc ^{ΔXF预处理动物}体内。尽管预处理诱导了显着的 ADA 反应，但我们发现 AvFc ^ΔXF的活性不受这些抗体存在的影响。这些结果表明，糖工程是增强 AvFc 抗肿瘤活性的有力策略。