- 作者：Xiaoniu Miao ; Andong Li ; Wei Chen ; Haidi Qi ; Zhen Qiu ; Yubin Zhang ; Juan Zhang ; juancpu@126.com ; Min Wang ; minwang@cpu.edu.cn
- 关键词：Human tumor necrosis factor ¦Á ; Functional antigen-binding fragment ; Single chain variable fragment
- 刊名：Biomedicine and Pharmacotherapy
- 出版年：2013
- 出版时间：June, 2013
- 年：2013
- 卷：67
- 期：5
- 页码：437-444
- 全文大小：1688 K
Aims
Single chain variable fragment (scFv) is one of the most popular recombinant antibody (rAb) formats. However, sometimes scFv with the most favorable specificity profile lack sufficient affinity or acceptable pharmacokinetics for clinical applications. To address these problems, we described a method to modify recombinant anti-rhTNF-¦Á scFv-F6D2E7.Results
Random mutations were inserted into CDR-H3 by performing PCR with tailored degenerate primers. After construction of a mutated antibody gene library, affinity selection was performed. Meanwhile the scFv (scFv-G10) selected from the library exhibited the most improved affinity to rhTNF-¦Á (2.9-fold higher than the parental scFv-F6D2E7). The scFv-G10 sequence and human constant (CH1 & CL) regions were used to construct a novel vector for developing an expression system that allows the production of a completely functional antigen-binding fragment (Fab) in Escherichia coli. The bioactivity of the Fab was determined by L929 cell cytotoxicity assay. Fab-G10 could neutralize rhTNF-¦Á-induced cytotoxicity to L929 cells, and the calculated 50 % inhibition rate (IC50) was 5.0 ¡Á 10?7 M.
Conclusion
We generated an artificial antibody fragment (scFv-G10) that had improved affinity and desirable specificity. Further, the Fab-G10 was constructed and expressed in E.?coli, where the bioactivity was further detected.