抗TNF-α/抗ED-B双特异抗体在酵母中的分泌表达与活性分析

Expression and characterization of a bispecific antibody targeting TNF-α and ED-B containing fibronectin

摘要:

赋予抗TNF-α 单链抗体片段 (TNF-scFv) 对炎症组织的特异性，用一段来自人清蛋白 (HSA) 的柔性连接肽在基因水平上连接TNF-scFv和抗B型纤维连接蛋白 (B-FN) 的额外域B (ED-B) 的scFv L19，构建了抗TNF-α/抗ED-B单链双特异抗体BsDb，其中B-FN为炎症组织中特异表达的抗原。BsDb在毕赤酵母中获得了分泌表达，表达产物经鉴定和纯化制备后，进行了功能分析。结果表明，BsDb保留了其亲本抗体TNF-scFv和L19对抗原的免疫反应性，能够同时结合TNF-α和ED-B，并中和TNF-α的生理作用。而且，BsDb对抗原的亲和力及中和能力与大肠杆菌包涵体来源的亲本抗体相比显著增强。在小鼠佐剂型关节炎 (AIA) 模型中，BsDb能选择性地积累和保留于小鼠的炎症关节，并快速从血浆中清除。说明BsDb兼备炎症组织的特异性和正常组织的低毒性，在类风湿关节炎及其他慢性炎症性疾病的治疗上具有较大潜力。

Abstract:

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.