title: Optimization and modification of anti-rhTNF-α single chain variable fragment antibody: effective in vitro affinity maturation and functional expression of chimeric Fab.
abstract: OBJECTIVE
  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-α 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-α (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-α-induced cytotoxicity to L929 cells, and the calculated 50% inhibition rate (IC50) was 5.0×10⁻⁷ M.
  
  
  CONCLUSIONS
  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.
authors.affiliation: School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Xiaoniu
authors.initials: X
authors.lastname: Miao
authors.forename: Andong
authors.initials: A
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Haidi
authors.initials: H
authors.lastname: Qi
authors.forename: Zhen
authors.initials: Z
authors.lastname: Qiu
authors.forename: Yubin
authors.initials: Y
authors.lastname: Zhang
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.forename: Min
authors.initials: M
authors.lastname: Wang
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D002460:Cell Line; D004926:Escherichia coli / Q000276:immunology*; D005347:Fibroblasts / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D051379:Mice; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014409:Tumor Necrosis Factor-alpha