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赋予抗TNF-α单链抗体片段(TNF-sc Fv)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-sc Fv和抗B型纤维连接蛋白(B-FN)的额外域B(ED-B)的sc Fv L19,构建了抗TNF-α/抗ED-B单链双特异抗体Bs Db,其中B-FN为炎症组织中特异表达的抗原。Bs Db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,Bs Db保留了其亲本抗体TNF-sc Fv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,Bs Db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,Bs Db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Bs Db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。
Given the specificity of the anti-TNF- [alpha] antibody fragment (TNF-sc Fv) for inflammatory tissues, a piece of flexible linker peptide from human albumin (HSA) is used to genetically attach the TNF-sc Fv and anti-B- Anti-TNF-α / anti-ED-B single-chain bispecific antibody Bs Db was constructed from sc Fv L19 of the extra domain B (ED-B) of protein (B-FN), which is specifically expressed in inflammatory tissues antigen. Bs Db was secreted in Pichia pastoris. The expressed product was identified and purified for functional analysis. The results showed that Bs Db retained the immunoreactivity of its parental antibodies, TNF-sc Fv and L19, against the antigen, bound both TNF-α and ED-B, and neutralized the physiological effects of TNF-α. Moreover, the affinity and neutralizing capacity of Bs Db for the antigen is significantly enhanced compared to the parent antibody derived from E. coli inclusion bodies. In the mouse adjuvant arthritis (AIA) model, Bs Db selectively accumulates and retains in the inflammatory joints of mice and is rapidly cleared from the plasma. It shows that Bs Db possesses the specificity of inflammatory tissues and the low toxicity of normal tissues, and has great potential in the treatment of rheumatoid arthritis and other chronic inflammatory diseases.