| Chimeric antigen receptors(Chimeric antigen receptors, CAR) is a fusion protein composed of a single chain antibody variable region and immune cells signaling molecules. CAR is expressed in the surface of effector cells(mainly immune cells) with a genetically engineered method, and then the immune cells are infused into the body. This treatment strategy is called CAR therapy. CAR therapy is a new adoptive immune effector cell therapy(ACT) strategy, involving redirecting immune cells, inducing them attack the tumor cells.Ad5F11p-GFP is serotype 5 adenovirus-based recombinant adenovirus, carrying the GFP reporter gene and Ad11 p targeting gene. In this study, three kinds of the immune cells, cytokine-induced killer(CIK) cells, primary natural-killer(NK) cells and natural-killer(NK) cells line NK-92, were infected by Ad5F11p-GFP at different multiplicity of infection(MOI), and uninfected immune cells were negative control. GFP expression was determined by flow cytometry; Cell morphology was photographed with microscope; cell proliferation was analyzed by trypan blue staining; Specific cytotoxicity of NK-92 cells was determined by LDH assay. Results: about 90% of infection efficiency could be achieved at 25 MOI on NK-92, while the infection efficiency for CIK was less than 40% at 200 MOI, and a lowest infection efficiency of primary NK cells. In addition, The infection efficiency basically unchanged at the same MOI between 48 h and 96h; and the immune cells infected with the virus were prone to agglomeration, displaying slowed proliferation, increased IFN-? release and strengthened tumor killing activity. Conclusion: Ad5F11p-modified NK-92 has a good prospect in adoptive immunotherapy(ACT).Wilms 'tumor gene(WT1) was the first discovered gene which is related to the occurrence and development of Wilms' tumor. The deletion of its allele function can lead to Wilms' tumor. WT1 expresses in a very narrow range, only in the fetal kidney, testes, ovaries and other tissues. It was also found that WT1 is highly expressed in leukemia cells and related to the occurrence development and prognosis of leukemia. In view of the above studies as well as a variety of excellent features an adenoviral vector, we want to build an adenovirus induction WT1-CAR system, and use it to modify NK-92 for the treatment of hematological malignancies. Methods: We designed a good specificity but not yet listed CAR, WT1-CAR. WT1-CAR is composed of WT1 recognition region, hinge region, CD28 inmembrane region, and CD28/41-BB/CD3 zete signal induction zone. Each element is separated by restriction sites for replacement components. For the convenience of making targeted peginterferon, Eco RI site is added after the signal peptide, and Bam HI site is coupled in the VH. As long as the Eco RI/Bam HI double digestion, it can be cloned into p IR-Ig G2N/IFN-Fc infer vector to form a long-acting interferon targeting. The WT1-CAR was loaded onto the p Shuttle-CMV plasmid, recorded as p Shuttle-CMV-WT1. After restructuring p Shuttle-CMV-WT1 and backbone plasmid Ad Easy-1/F1 lp, it was inferred to HEK293 cells with Lip2000 to package viral agents. Then the recombinant adenovirus were identified gene and protein levels. Finally, they are amplified and purified. Results: We successfully packaged recombinant virus Ad5F11 p expressing WT1-CAR fusion gene, named as Ad5F11p-WT1/CAR. However, we found that when HEK293 passaged 7-9 times at the amplification of recombinant viral, the infection efficiency on HEK293 drastically reduced, and the virus titer also correspondingly reduced after purification. Conclusion: The adenoviral vector Ad5F11 p can be used to induce WT1-CAR gene, and it has a good prospect in the CAR project. |
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