Nanoparticles Conjugation Anti-OX40mAb/anti-AFP MAb Induction CTL Cell Antitumor Responses On Hepatocellular Carcinoma

Objective：1. Prepare nanoparticle by using biocompatible and biodegradable PLGAand couple anti-OX40and anti-AFP antibodies.2. To observe the cytotoxic Tlymphocyte (CTL) biological effect of the anti-OX40and anti-AFP nanoparticles.Methods:1. Using PLGA、acetonitrile and F-127to prepare PLGA nanoparticles onthe basis of deposition. And couple anti-OX40and anti-AFP antibodies on the surfaceof PLGA nanoparticles with NHS and EDC.2. Isolate peripheral blood mononuclearcells, then separate and pure CD4+and CD8+cells by VarioMACS. Amplify anddifferentiate CD14+into DC cells with “IL-4+GM-CSF”, use mitomycin to inactiveDC cell,then mix with CD8+cells in proportion to cultivate CTL cells.3. Theproliferation and the release of IL-2and IFN-γof CTL cells for anti-OX40andanti-AFP antibodies onto PLGA nanoparticles measured by WST-1assay and ELISAassay when CTL cells were induced by anti-OX40and anti-AFP antibodies ontoPLGA nanoparticles.4. The cytotoxicity of CTL cells against a hepatocellularcarcinoma cells were measured by LDH assay.Results:1. Observed the nanoparticle in scanning electron microscope, the PLGAnanoparticle is an uniform circular particle size.average size is396nm, The zetapotential of PLGA nanoparticle is-(35.12±5.34)mV.2. Using VarioMACS can gethighly pure CD14+and CD8+cell, highly pure CD14+cells can differentiationmature DC cells.3. Anti-OX40/anti-AFP nanoparticles can induce CTL cellsproliferation and activation，the proliferation index is (151.49±16.25)%，secretion ofIL-2and IFN-γis（130.53±37.058）pg/ml、（265.37±17.05）pg/ml。4. Usingfluorescence microscope to observe fluorescent membrane staining cell CTL cells andHepG2cells,when adding Anti-OX40/anti-AFP-Nanoparticles, CTL cells andHepG2cells were tightly bonded,and much more cells combine closely. Conclusion: Anti-OX40/anti-AFP-Nanoparticles can stimulate CTL cellsproliferation and activation, and Anti-OX40/anti-AFP-Nanoparticles can be moreclosely integrated between the CTL and HepG2cell,enhanced CTL cells killingeffect on HepG2cells.LDH analyses showed that the anti-OX40-PLGA nanoparticlesgroup mediated CTL cytotoxic activity against hepatocellular carcinoma cells wasincreased significantly （P＜0.05）.