基于TNF功能表位设计新型人源单链抗体

目的开发基于TNF功能表位的新型人源单链抗体。方法利用自主研制的鼠抗TNF-α中和单抗Z12能特异性识别TNF-α的141~146位功能表位特性,通过理论模拟构建TNF/抗体Z12相互作用的复合物模型设计获得功能性拮抗肽(PT2、PT3、PT4、PT7)以及单域抗体PTVH5(以人抗体可变区重链框架VH5为支架合理展示PT2、PT3、PT4),利用计算机辅助分子设计以及同源模建、分子对接方法,进一步合理选择人抗体可变区轻链框架(Vκ1)作为展示支架,通过构象判别、作用能比较以及识别区域确认并选择合适的连接肽设计新型单链分子ScFv_AB1。结果理论分析发现,ScFv_AB1稳定性较好,识别TNF-α的141~146功能位点(即Z12识别的位点)。生物学实验证明,ScFv_AB1能与TNF-α结合、抑制TNF-α与TNFR的结合、抑制TNF-α介导的细胞毒作用。结论初步验证了"借助计算机模建,基于人抗体可变区的框架结构和拮抗肽设计单链抗体分子"的策略是可行的,从而为人源小分子抗体的制备提供了一条可供选择的途径。

Novel human single chain antibody design based on the functional epitope of TNF

Objective Developed novel human single chain antibody which design based on the functional epitope of TNF. Methods In a previous study, we obtained a TNF-α neutralizing mAb Z12 and identified the epitope （i.e. from 141 to 146 aa in TNF-α） recognized by Z12. Moreover, we explored a series of TNF-α antagonist peptides （i.e. PT2, PT3, PT4 and PT7） and the single domain antibody PTVH5 （using human antibody frame work of variable region of heavy chain, i.e. VH5, as scaffold to display the peptides PT2, PT3 and PT4） based on the interaction between TNF-α and mAb Z12. In the present work, based on the computer-guide molecular design, homology modeling and molecular docking method, we used human antibody frame work of variable region of light chain （Vκ1） as dispalying scaffold and obtained a novel single chain antibody, named as ScFv_AB1, according to the conformation, binding energy and identified domain. Results Theoretical analysis showed that ScFv_AB1 was stable and recognized the position 141-146 of TNF-α （same as Z12）. Biological experiments showed that ScFv_AB1 could bind to TNF-α, competitively inhibit the binding of mAb Z12 to TNF-α, block the binding of TNF-αto TNFR and inhibit TNF-induced cytotoxicity. Conclusion This study demonstrated that it is feasible to design novel single chain antibody based on human antibody consensus frameworks and antagonist peptides using computer-guided modeling method. It also provides an alternative way to obtain human small molecular antibody.