Nature Killer Cells Modified By Tls11a-Fc Bifunctional Complex Target Hepatocellular Carcinoma Specifically

Background:Malignant tumor,a kind of deadly disease,affects human health seriously and has become one of the main causes of death.It has been about 30 years since the NK cells used for the immunotherapy of tumor.It has attracted the attention of people from then on,but the development of NK cells in clinical applications is slow and still unable to make breakthrough.Therefore,making research on how activating NK cells effectively and improving its killing effect is significant.Furthermore,this study may provide a new method for the immunotherapy of tumor.Objective:Synthesis a bifunctional complex based on aptamer and Fc protein,which not only specifically bind to tumor cells,but also bind to NK cells,to enhance anti-tumor effect.Methods:Use the Sulfo-SMCC crosslinker to construct a bifunctional complex TLS11a-Fc.The synthetic complex was measured by SDS-PAGE gel electrophoresis.The connection efficiency of aptamer was examined by fluorescent photometric analysis meter.The toxicity of bifunctional complex to cells and tissues was determined by MTT test and HE experiment.The high purity of CD56~+CD16~+NK cells was sorting out by using magnetic bead separation.The binding ability of the bifunctional complex TLS11a-Fc to NK cells and hepatocellular carcinoma cells was examined by flow cytometry and fluorescent microscope.Utilize the flow cytometry to measure the expression of CD107a and CD69 on the surface of NK cells.The secretion of TNF-αand IFN-γwas investigated by ELISA and ELISPOT.The killing effect of NK cells to target cells was detected by flow cytometry.In vivo,the activity of NK cells modified by TLS11a-Fc complex against tumor was investigated in nude mouse liver cancer xenograft model.The model was established by injecting hepatocellular carcinoma cells subcutaneously.Mice then were intravenously injected via tail vein with different formulations.Results:Bifunctional complex was synthesized successfully.The connection efficiency of aptamers in complex was above 75%.Bifunctional complex was non-toxic to the tissues and cells in vivo or vitro.The separation efficiency of CD56~+CD16~+NK cells was above 90%.The complex has the characteristics of binding to NK cells and hepatocellular carcinoma cells.The modified NK cells expressed CD107a and CD69 increasingly,as well as secreted TNF-αand IFN-γcytokines.NK cells modified by bifunctional complex have a specific lysis against liver cancer cells in vitro.NK cells modified by bifunctional complex can prolong the survival time of mice,inhibit tumor volumes,inhibit the proliferation of tumor cells,and promote tumor cell apoptosis.Conclusion:The bifunctional complex TLS11a-Fc modified NK cells can target hepatocellular carcinoma cells specifically,which may provide a novel method to realize the immunotherapy for patients with hepatocellular carcinoma or other tumors.That has a great prospect for clinical applications.