Expression Of Recombinant Immunotoxin Against Human Glioma, SZ39(ScFv)-PE40, And A Humanized Anti-CD3 Single-chain Fv Fragment In Escherichia Coli

A recombinant immunotoxin against human glioma, SZ39(ScFv)-PE40, was constructed by fusing the cDNA of SZ39-ScFv, which was demonstrated to have the capacity of specifically binding to glioma cells in our previous study, to a truncated form of Pseudomonas exotoxin A without its binding domain (PE40). High level expression was achieved in E.coli, and the primary study demonstrated that SZ39(ScFv)-PE40 possessed the binding activity to glioma cells in vitro, which warrants a further study on the anti-glioma recombinant immunotoxin. In another part of present study, a humanized anti-CD3 single chain Fv fragment (ScFv) was constructed and expressed, which was prepared as a part of an anti-CD3/anti-glioma bispecific antibody (BsAb) with low immunogenicity and small size.Part I. Construction and Expression of a Humanized Single-chain Fv Fragment in E.coliObjective: In previous study, the anti-CD3/anti-glioma BsAb prepared by chemical conjugation was demonstrated to have specific anti-glioma effects in vitro and in animal models, but the molecular size of the BsAb was too big and its preparation was somewhat difficult. Thus, in present study, we constructed and expressed a humanized anti-CD3 single chain Fv fragment(ScFv), which would be a promising candidate for the construction of a recombinant anti-CD3/anti-glioma BsAb with small size and more potency. Methods: The cDNA encoding the variable regions of a humanized anti-CD3 antibody, namely the VH and VL genes were amplified by PCR in which plasmid pZZ3 served as template, then the VL and VH genes were attached to the oligonucleotides encoding the linker (Gly4Ser)3 to construct the anti-CD3-ScFv gene, which was inserted into prokaryotic expression vector pET20b(+) and pGEX-5X-1 respectively to construct expression vector pET20b(+)-anti-CD3-ScFv and pGEX-5X-1-antiCD3-ScFv, and then pET20b(+)-anti-CD3-ScFv was...