Construction Of PD-1 ScFv-Transforming Lactobacillus And The Therapeutical Effects On Tumors-Bearing Mice

BackgroundCytotoxic T lymphocyte is specific T cell.It have characteristic of effective targeted killing of tumor cell.Immune checkpoint PD-1(programmed cell death protein 1)discovery,which provides a new idea for improving the efficacy of CTL.PD-1 is negative regulator proteins on T cell activation which can inhibit the activity of T cells and reduce the antitumor function of T cell.The cholera toxin B subunit is the regulatory subunit that could be recognized and binded by intestinal epithelial cells,and it could specifically combine with the Ganglioside M1 receptor,had no enterotoxigenic effects and a mean to improve the mucosal immunity of oral vaccines.Lactobacillus is an important probiotic in human and animal gut with various functions,its retention time is longer in the lower digestive tract and with large number,which is conducive to the expression and absorption of peptide drugs and suitable for intestinal expression and administration carrier.ObjectivesThe recombinant human soluble PD-1ScFv fusion protein was expressed with food grade lactic acid bacteria as the host,and analyzed its tumor immunotherapy.MethodsThe CTB-hsPD-1ScFv gene expression vector LZ-CTB-hsPD-1ScFv and the hsPD-1ScFv expression vector LZ-hsPD-1ScFv were constructed by molecular cloning technique as the experimental control to analyze the effects of CTB.Transformation of Lactococcus was conducted by electroporation.In vitro experiments:Select the best induction conditions of transformant bacteria,analyze the endocytosis and its stimulating effects to the mice spleen lymphocyte activation level of fusion protein cell.In vivo:the B16F10 tumor mice model were used to analyze its effects to the pathological changes of the tumor,the activation level of spleen T lymphocytes to confirmed the therapeutic effects of CTB-hsPD-1ScFv transformed bacteria.Results(1)The pLZ-CTB-hsPD-1ScFv and pLZ-hsPD-1ScFv were successfully constructed.(2)We successfully obtained the LZ-CTB-hsPD-1ScFv and LZ-hsPD-1ScFv.(3)LZ-CTB-hsPD-1 ScFv could express fusion protein in vitro and could be endocytosed by intestinal epithelial cells mediated by CTB and release the hsPD-1ScFv to activate T cells and to improve the efficacy of CTL.(4)LZ-CTB-hsPD-IScFv could be induced in the mice intestine after feeding,and could be absorbed and transformed by intestinal epithelial cells,and release the hsPD-1ScFv into the blood.(5)In vivo treatment,LZ-CTB-hsPD-1ScFv showed significant therapeutic effects on melanoma transplanted tumor,and could prolong the survival time of tumor-bearing mice,inhibit tumor cell proliferation,inhibit the tumor angiogenesis,and induce the tumor tissue apoptosis.(6)It was proved that CTB-hsPD-1ScFv fusion protein showed better immunostimulatory effects on spleen lymphocytes.ConclusionWe successfully constructed the recombinant human soluble PD-1ScFv lactic acid bacteria.And it could regulatablely secret the CTB-hsPD-1ScFv fusion protein in the intestine,and could be absorbed by intestinal epithelial cells to improve the activated level of T lymphocyte,improve the immune system,prolong survival,inhibit tumor growth and induced apoptosis,showing significant therapeutic effects to the tumor.