Efficient Induction Of Antitumor Immunity By Tumor Cell-Derived Exosomes Modified By Anchorage Of Superantigen SEA

Exosomes are small vesicles with endocytic origin secreted by many live cells. They are flattened or round sphere sequestered by a lipid bi-layer visualized by under electron microscopy (EM), with sizes from 30nm to l00nm. Though the discovery of exosomes could be traced back to 1987, only did recent years the exosomes attract much more attention by the immunologist and oncologist since Zitvogel L et al found that dendritic cell-derived exosomes can eradicate established murine tumors in 1998. Up to now, extensive studies have been conducted about the preparation, component, biogenesis and functions of exosomes. It have been found that many cells can secrete the exosomes, such as dendritic cells (DC), cytolytic T cells (CTL), EBV-tansformed B cells, mast cells, platelets, mastocytes, epithelial cells and tumours of haematopoietic or non-haematopoietic origin.By using DC-derived exosomes (DEXs), the phase I clinical trial conducted by Duke University Medical Center andImmunotherapy Department of Institut Gustave Roussy independently have accomplished recently, and the patients included stage III/IV melanoma patients and advanced non-small cell lung cancer patients. Those results demonstrated that production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC and melanoma. Some patients experienced long term stability of disease and activation of immune effectors. But, as we knew, DC culture is time-consuming, tedious, expensive, and difficult to be cultured to a large amount. So, it needs to find other cell resources to produce the exosomes. Tumor cells or EBV-transformed human B lymphcytes are optimal exosomes-producing cells, which can be easily cultured and less expensive with more potential application to clinic.To build a platform for large-scale exosome preparation, we selected the EG7 cells as the exosome-producing cells. EG.7 cells, which are EL4 cells transfected with OVA gene, can secrete OVA. As a suspending cell line, EG.7 can be cultured in a spinning flask, and only one flask with the volume of 500 ml can expand 109 cells within less than 10 days. To shorten the preparation time, we used the 3/0.8 Am Sartoclean CA filter to discard the cells, debris and large gradules. To concentrate the clarified supernatants, we used 3×500-kDa MWCO hollow fiber membrane cartridge (Biomax 500, each has working area of 50 cm²) in Labscale TFF System (Millipore) to ultracentrifuge them to the final volume less than 50 ml. Then the samples were floated onto a density cushion composed of 20 mM HEPES/NaOH (pH7.2.) sucrose /30% sucrose/45% sucrose and ultracentrifuged at 100,000×gand 4℃ for 2h in a SW-32 swinging bucket rotor. To achieve approximately 97% removal of the sucrose, the sample was diafiltered with a minimμm of five volμmes of PBS. For in vivo experiments, the samples were passed through a 0.22-μm Microcentrifuge Filters (Sigma) to get the sterile exosomes.We have visualized the purified exosomes with high purity under the EM. those particles with the sizes at 40-100nm, with clear bi-lipid membrane. And, those exosomes richly contained HSP, HSC and OVA as demonstrated by Western blot. As the sizes of exosomes are less than 100nm, currently, there are two methods to calculate the size of the nanoparticle, one is the electronmicroscope, another is the size determination by Zetasizer. The former not only can visualize the sizes and also the structure of the particles. But the EM results don't faithfully demonstrate the particle size distribution, on the contrary, the Zetasizer can express the size distribution on the whole and with statistical significance. Finally, due to the model antigen OVA secretion by the EG.7 cells, we calculate the OVA amount as a more direct method to quantitate the exosomes by the absorption ELISA. There were about 1-5×10¹³ molecules of OVA in exosomes derived from l×10⁹ EG.7 cells.As a cell-free cancer vaccine, exosomes either derived from DCs or tμmor cells have anti-tμmor activities, but far from what had expected initially.