HS83P34,an Activated T Cell Selective Deletion Molecule: Construction, Preparation And Function

The rate of acute rejection in clinical transplantation has been decease and the rate of the long-term or short-term survival has been increased due to the application of immunosuppressors clinically. However, the side effect of long-term using immunosuppressor may lead to chronic pharmic injuries and restrain more increase of long-term graft survival rate. It is important problem in present on how to specifically inhibit the immunoreaction while insuring long-lasting function-graft survival and improving the quality of patients' life. So it is a key point for the research to design a new specific immunosuppressor, which should be higher efficiency, lower toxin, lower infection and lower tumor risk.As activated T cells playing a central role in transplantation rejection, it is a major goal of transplant immunotherapy to them. Activated T cells express a number of markers including CD25 (IL-2 receptor α), cytotoxic T lumphocyte-associated Ag-4 (CD152), CD69, CD147. The antibodies of these markers can be good guided moieties of immunotoxins intending forselectively depleting activated T cells.The majority of immunotoxins studied to date toxins act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as pore-forming protein. Perforin is such a protein.In the studies, we describe the genetic recombination, preparation, and the in vitro and in vivo results obtained with an immunotoxin consisting of perforin, linked to recombinant humanlized mAb against CTLA-4, generated by selecting a synthetic phage single-chain fragment variable (scFv). Fv fragments are the smallest functional units of Abs required to maintain the binding and the specificity of the whole Ab. Fragment variables can be produced as single chains (scFvs) by recombinant techniques. In this article, we recombinant, purify and produce hS83-P34, study in vitro results about specificity, safety, sensitivity to T cells, and in vivo effect in rejection model. The results were as followed:The genes of anti-CTLA-4-hS83 and pore-forming domain of perforin were amplified via PCR, and legated the two fragments together using T4 DNA ligase. The successfully constructed plasmid was named as pBS-hS83-P34. Double enzymes digest the pBS-hS83-P34, and then cloned into expression vector pQE30. Transfered the successfully constructed plasmid pQE30-hS83-P34 in E.coli M15 to express target protein hS83-P34, and monitored hS83-P34 expressing as DBS.Collected the inclusion bodies (IBS), washed them twice with IBS washing buffer for 1 hour at 30℃, and then dissolved the IBS with dissolving buffer overnight at 4℃. Next day, purified the denatured protein withDEAE-Sephedex Fast Flow for further purification. The target protein hS83-P34 concentration was more than 95% in penetration peak. So the purified protein was refolded by dialysis buffer at 4℃ for 48 hours to get the refolding recombinant immunotoxin hS83-P34.The in vitro toxicity of hS83-P34 was tested using cell models. The target cell was CTLA-4 positive cell lines Raji, CEM, and activated T cells, and the endothelial cell line (ECV304) and unstimulated T cells were used as negative control cells. Protein hS83 was negative control and PBS was blank control. Added the immunotoxin in cell culture firstly, then monitored the cells number with MTS at 490nm. The livability of PBS blank group was seemed as 100% to calculate the livability of cells. Those results were showed: First, hS83-P34 specifically combined and killed the CTLA-4 positive cells with high efficiency. 20μl hS83-P34 (final concentration was 1.0μmol/L) was added in cell culture of activated T cells, CEM and Raji, the livability reduced at 8 hours, reduced lowest at 18 hours, and then lasted 36 hours. Secondly, hS83-P34 had lower toxicity to normal endothelial cells or naive T cells. 20μl hS83-P34 (final concentration was 1.5μmol/L) added in cell culture 24hours, the livability of ECV304 and unstimulated T cells was higher than 85%, while which of the CTLA-4 positive cells was only about 15-20%. Lastly, the two parts of the recombinant immunotoxin hS83-P34 are all humanlized and have a lower molecular weight than whole molecules. These features might give low heterogeneity to this recombinant protein.To preliminarily verify the in vivo availability and activity of hS83-P34, we used a mouse model consisting of implant allogeneic tumor cells, capable of giving an acute immune rejection with a T lymphoid infiltrate as describedby Garlanda et al. Briefly, Balb/c and C57BL/6 are different lines, they have different MHC molecule. B16 cell line from melanin tumor was derived from C57BL/6 mice, which hold same MHC with C57BL/6. These cells are able to elicit an allogeneic response when injected in Balb/c mice, with a picture comparable with an acute rejection showing a T lymphoid cell inflammatory infiltrate. The B16 cells are quickly killed, while an immune suppression could diminish the reaction and prolong the survival of the tumor cells.B16 cells (5×106) were injected s.c. into Balb/c front leg at day 0. The experiment group of 12 mice was injected hS83-P34 (12mg/kg) into abdomen. As a control, a group of 12 mice was injected salt water with same schedule. Animals were sacrificed at day 4 or day 13. Tissue corresponding to the site of injection was fixed in 10% formaldehyde, processed for light microscopy and examined T cells by immune fluorescene. The primary results in vivo experiment of hS83-P34 showed that hS83-P34 decreased the T lymphocytes in graft, decreased the transplantation rejection and prolonged the survival of graft.From the results, we can see that we successfully constructed a recombinant immunotoxin using anti-CTLA-4 scFv as target fragment and a pore-forming domain of perforin with 34 amino acids as killing fragment. hS83-P34 possesses the features of immunotoxin: specificity, killing activity, low toxicity to normal cells. It can kill activated T cells and prolonged the survival of graft. So it maybe have good prospect for prevention and cure ofrejection.