[In vitro molecular evolution of humanized mouse ScFv specific against human cross-linked fibrin]

In early work at our laboratory, a mouse ScFv All specific against human cross-linked fibrin was picked out by using phage displaying technique, and it was useful in prepareing targeting thrombolytic agent. To reduce its immunogenicity, All was humanized according to the surface reshaping approach. In order to increase the affinity and specifity of humanized ScFv, the Mixed HCDR3 and LCDR3 library was constructed and selected, and five different humanized ScFv with better affinity or specifity than humanized ScFv were identified, but the affinity of these five humanized scFvs was still lower than parent mouse scFv All. In this study, in order to further increase the affinity of humanized scFv, the identified five different CDR3 mutant of humanized ScFvs were mutated and reassembled with the methods of error-prone PCR and DNA shuffling, then the shuffled ScFvs were cloned into plasmid pHB-1 HSCFV to construct ScFv library of 10(5). The ScFv library was displayed on the surface of phage and panned with antigen DD, then five ScFvs with better affinity or specifity were identified by using ScFv-alkaline phosphatase detection system which was established in our laboratory. Based on identified five better ScFv, the second round error-prone PCR and DNA shuffling program was taken out, a ScFv library of 10(6) was constructed and selected as before. And four humanized ScFv that had evidently better affinity than parent mouse ScFv was identified. This work laid a foundation for further research of targeting thrombolitic agent with low immunogenicity.