| Organ transplantation has become the best treatment for end-stage organ diseases.However, rejection remains one of the major obstacles for the survival of allograft.Chemical immunosuppressant can suppress rejection, but associated with significantside effects, which jeopardized its clinical application. Biological immunosuppressiveagents, especially immunotoxin, can clear antigen-specific T cells through its specificbinding with less side effects and better prospect for clinical application.An immunotoxin is a human-made protein that consists of a targeting portion (vector)linked to cytotoxic molecules (toxin). When the protein binds to that cell, it is taken inthrough endocytosis, and the toxin kills the cell. It has been reported in the treatment ofcancer and transplant rejection.CTLA-4is a molecule that only expressed on the surface of activated T cells but noton stationary T cells. Therefore, CTLA-4antigen binding molecule is an idealimmunotoxin vector. Immunotoxins generated by these vectors only kill activated Tcells and tumor cell, avoiding non-specific damage to normal stationary T cells.Melittin (Mel), the main component of bee venom, is an important antimicrobialpeptide with antibacterial, anti-radiation, anti-tumor effects. It is composed of26aminoacids. Melittin has low molecular weight, low immunogenicity, and low susceptibilityto allergic reactions. Its membrane activity can directly dissolute the phospholipidmembranes of cells, inhibit the development of cell. Melittin is an ideal toxin fragment.In this study, we constructed a humanized anti-CTLA-4single-chain antibody as atargeting fragment, which can specifically bind to the activated T cell expressingCTLA-4. Melittin analogues were used as the toxin fragment to specifically killactivated T cells. The immunotoxin molecule prepared in our study displayed highefficiency and low toxicity that could be used for anti-transplant rejection. Using the overlap extension PCR technique, recombinant immunotoxins full-lengthcDNA were obtained by two-step PCR. Then connected with the expression vectorpBV220, and transformed into Escherichia coli. The recombinant gene was expressed asinclusion bodies in Escherichia coli expression system. Inclusion bodies weredenaturized with8M urea, renatured by the dilution method, and then purified usingSP-Sepharose Fast-Flow cation exchange column. After chromatography purificationthrough Sephacryl S-200gel filtration column, the target protein CTLA4-ScFv-Mel wasprepared.In the in vitro activity study, non-activated T lymphocytes, negative control ECV-304cells, T lymphocytes activated by ConA, and CTLA-4positive Raji cells and Jurkatcells were treated by2μmol/L of CTLA4-ScFv-Mel. The results showed that the cellsurvival decreased24h after administration of CTLA4-ScFv-Mel. The survival rates ofthe first two groups were78.9%and77.3%, respectively; while the survival rates of thelatter three groups were31.2%,32.7%and30.8%, respectively. The results suggestedthat CTLA4-ScFv-Mel has higher destruction rate in CTLA-4positive cells than that innormal cells, showing its selectivity and high efficiency in cell killing.BEL-7402liver cancer xenograft and S180mouse xenograft tumor model were usedin the vivo activity study. CTLA4-ScFv-Mel were given in low, middle, and high dose.Pharmacodynamic indicators including tumor xenografts relative volume was comparedbetween the test group and negative control group. The results showed thatCTLA4-ScFv-Mel can promote the survival and growth of transplanted tumors in thetwo test groups, presenting a certain dose-effect relationship. The inhibition of T cellimmunity was suggested to be related with this phenomenon.This study has successfully prepared a new immunotoxin fusion protein, andevaluated its initial activity and pharmacodynamic properties, providing a potential drugfor the treatment of organ transplant rejection with good scientific value anddevelopment prospects. |
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