Molecular construction and characterization of a novel exotoxin fusion protein that selectively blocks the B7:CD28 costimulatory signal system.

An important strategy for specifically preventing and treating graft-versus-host and host-versus-graft diseases is to selectively block the B7:CD28/cytotoxic T-lymphocyte A4 costimulatory signal system for induced immune tolerance. In this study, a novel recombinant B7-2-L-PE40KDEL fusion protein was created to target the B7:CD28 system. We used a flexible linker sequence (Gly4Ser)4 and overlapping sequence extension to link the cDNAs encoding a human B7-2 extracellular domain and a mutant truncated form of Pseudomonas exotoxin A (PE), PE40KDEL. This B7-2-L-PE40KDEL fusion gene was then inserted into the pTYB4 expression vector, expressed in Escherichia coli, and purified through Ni-NTA mealty affinity-->MonoQ anion exchange-->Superdex75 gel filtration chromatography 3-step purification protocols. Western blotting demonstrated that the B7-2-L-PE40KDEL fusion protein specifically bound antihuman B7-2 monoclonal antibody and anti-pseudomonas exotoxin A antiserum. We used the Antheprot nucleic acid and protein analyzing software to predict the characteristics of this fusion protein, and showed that the fusion did not confer new antigenicities to the fusion protein. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests demonstrated that at doses ranging from 0.2 to 2 microg/mL, this fusion protein specifically killed CD28-overexpressing Jurkat cells but even at doses of 2 microg did not kill CD28-negative Hut28 cells. The results of a one-way mixed lymphocyte reaction demonstrated that the fusion protein has a range of suppressive effects on HLA class I and II matched related donors and recipients, and HLA class I and II mismatched unrelated donors. Taken together, these results demonstrate that we have developed a novel recombinant human B7-2-L-PE40KDEL exotoxin fusion protein that specifically blocks the B7:CD28 costimulatory signal system in a manner that may be of significant importance in preventing and treating graft-versus-host or host-versus-graft diseases.