Functional production of human antibody by the filamentous fungus Aspergillus oryzae
Functional production of human antibody by the filamentous fungus Aspergillus oryzae
复制标题
DOI：
10.1186/s40694-020-00098-w
复制
发表时间：
2020-05-28
影响因子：
--
通讯作者：
Maruyama, Jun-ichi
中科院分区：
文献类型：
--
作者：
Huynh, Hung Hiep;Morita, Naoki;Maruyama, Jun-ichi
背景：作为生物药物的单抗在当前的治疗应用中发挥着关键作用。通常，哺乳动物细胞系，如来自中国仓鼠卵巢(CHO)的细胞系，被用来产生重组抗体。然而，人们仍然担心使用哺乳动物细胞时的高昂成本和致病污染的风险。米曲霉是一种丝状真菌，被公认为GRAS(通常被认为是安全的)生物体，能够向培养上清液中分泌大量蛋白质，因此该真菌已被用作生产异源蛋白的经济有效的微生物宿主之一。为了实现这一策略，世界上最畅销的用于治疗包括类风湿性关节炎在内的免疫介导性炎症性疾病的抗体之一--人类抗肿瘤坏死因子α抗体adalimumab--被米曲霉选为生产全长mAbs。通常情况下，抗体的N-糖基化通过与免疫细胞上的Fc受体(Fc-Gamma R)结合来影响免疫效应功能，如抗体依赖的细胞介导的细胞毒作用(ADCC)。利用CRISPR/Cas9系统首先删除了真菌高甘露糖化过程中的关键酶Aooch1基因，以研究抗体与Fc-Gamma RIIIa的结合能力。结果：利用α-淀粉酶AmyB融合蛋白系统在米曲霉中表达了阿达利单抗。在培养上清液中成功制备了由两条重链和两条轻链组成的全长阿达利单抗。在产生抗体的菌株中，10-蛋白酶缺失菌株的抗体效价最高(39.7 mg/L)。采用蛋白A和体积排阻层析两步纯化，得到高纯度的样品用于进一步分析。米曲霉生产的adalimumab的抗原结合和肿瘤坏死因子α中和活性与商业产品Humira(R)相当。即使用Aooch1缺失菌株改变N-糖链结构，重组adalimumab也没有检测到与Fc-Gamma RIIIa的明显结合，这表明免疫效应功能只有很小的额外活性。结论：这些结果为利用米曲霉生产人类抗体提供了一种替代的低成本平台，可能为患者的福利提供了合理的支出。
Background: Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNF alpha antibody adalimumab, one of the world's best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (Fc gamma R) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with Fc gamma RIIIa. Results: Adalimumab was expressed in A. oryzae by the fusion protein system with alpha-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNF alpha neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira (R). No apparent binding with the Fc gamma RIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions. Conclusion: These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient's welfare.