Lai Huafang, He Junyun, Hurtado Jonathan, Stahnke Jake, Fuchs Anja, Mehlhop Erin, Gorlatov Sergey, Loos Andreas, Diamond Michael S, Chen Qiang
The Biodesign Institute, Arizona State University, Tempe, AZ, USA.
Plant Biotechnol J. 2014 Oct;12(8):1098-107. doi: 10.1111/pbi.12217. Epub 2014 Jun 29.
Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ∆XF plants, a glycosylation mutant lacking plant-specific N-glycan residues. Glycan analysis revealed that ∆XF plant-derived pE16scFv-CH (∆XFpE16scFv-CH) and pE16 (∆XFpE16) both displayed a mammalian glycosylation profile. ∆XFpE16 and ∆XFpE16scFv-CH demonstrated equivalent antigen-binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell-produced E16 (mE16). A single dose of ∆XFpE16 or ∆XFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost-effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases.
此前，我们团队构建了一种植物源单克隆抗体（MAb pE16），其能有效治疗小鼠的西尼罗河病毒（WNV）感染。在本研究中，我们开发了一种pE16变体，它由一个单链可变片段（scFv）与人IgG的重链恒定区（CH）融合而成（pE16scFv-CH）。pE16和pE16scFv-CH在本氏烟草∆XF植物（一种缺乏植物特异性N-聚糖残基的糖基化突变体）中高效表达并组装。聚糖分析显示，源自∆XF植物的pE16scFv-CH（∆XFpE16scFv-CH）和pE16（∆XFpE16）均呈现哺乳动物糖基化特征。∆XFpE16和∆XFpE16scFv-CH表现出同等的抗原结合亲和力和动力学，并且与亲本哺乳动物细胞产生的E16（mE16）相比，在体外对WNV的中和作用略有增强。单剂量的∆XFpE16或∆XFpE16scFv-CH即使在感染后4天也能以与mE16相同的比率保护小鼠免受WNV诱导的死亡。本研究对治疗性单克隆抗体及其在糖工程植物中产生的单链变体的表达、结构和功能进行了详细的串联比较。此外，它还展示了抗WNV单克隆抗体治疗变体的开发，这些变体在疗效上与pE16相当，生产更简单，并且由于其哺乳动物N-糖基化，作为治疗剂使用可能更安全。该平台可能会导致针对WNV感染以及其他传染性、炎症性或肿瘤性疾病的基于抗体的治疗剂的更强大且更具成本效益的生产。
