从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础

Isolation and Characterization of Anti-TNF Peptides from Phage-displayed CDR-based Peptide Libraries

As complementarity determining regions (CDRs) of antibody could mimic both specific binding to antigen and biological properties, peptide analogues of CDRs can be developed. Corresponding to the deduced CDRs amino acid sequences of cA2, six CDRs based peptide libraries were constructed using phage display technique. cA2 is a chimeric (human mouse) monoclonal antibody against human tumor necrosis factor (TNF) that consists of the variable region of murine anti TNF mAb coupled to the constant region of human IgG1κ. CDR based peptide sequences included amino acid sequences from the CDRs of the Ab and the three random residues flanking CDRs (X3 CDR X3). Phage clones were selected on rh TNFα coated microplate. Clones of phage peptide were obtained and further tested by ELISA. Seven CDR derived peptides were isolated (termed CDR2L1,CDR2L 2,CDR2L 3, CDR1L1, CDR2H1, CDR3H1,CDR3H 2) and showed TNF binding capacity. The activity of inhibiting human recombinant TNF alpha (rhTNF α) was detected on L929 cell using MTT method. The data show that CDR2L2 and CDR3H2 phage peptides can inhibit 38% and 20 7% of TNF alpha activity, respectively when TNF alpha gives 57% cell toxicity on L929 Moreover, CDR2L2 phage peptide of all peptides was most effective in inhibiting TNF induced cytotoxicity on L929 cells. The results indicate that the information obtained from a novel exploration of anti TNF peptide antagonists from CDRs of antibody variable sequences may become a potential identification method in pharmacology. The method could be of interest for the rational identification of biologically active peptides derived from CDRs of therapeutic antibody.