Targeting Tissue Factor On Tumor Vascular Endothelia Cells And Tumor Cells For Immunotherapy

The definition of tumor gene therapy is importing the functional gene to the cancer cells or tissues and use its gene product to kill the cancerous cells. How to build a safe and effective tumor target gene therapy is the research hotspot. The over-expression of tissue factor (TF) observed in many malignant tumor cells and tumor vascular endothelial cells except normal vascular endothelial cells make TF an ideal target for cancer therapy. Farctor Ⅶ is a kind of thrombin that can specific bind to the TF, It is composed of 20 kD light chain and 30 kD heavy chain which are linked by a disulfide bond. The light chain is responsible for bond to TF while the heavy chain intiates the blood coagulation pathway, Because mouse light chain of FVH(mLFⅦ) binds strongly both to human TF and mouse TF,unlike human light chain of FⅦ (hLFⅦ) that binds strongly to human TF but weakly to mouse TF. Therefore, the mLFⅦ can be used in tumor therapy as a tumor target molecule. IgG is a kind of immunoglobulin that widely distributed in the body and has high concentration in blood and extraceller fluid. The amino-terminal variable domain called Fab which can bind to antigen and the carboxy-terminal constant domain called Fc which can bind to cells express Fc receptor (FcR). In such way, the antigen(such as tumor cells,virus or bacterial infected cells) can be eliminated by FcR. Fab bind to antigen is specific while Fc bind to FcR is not specific. Therefore,we can use tumor target molecule instead of Fab and keep Fc to construct a new protein which can active immune system and kill tumor cells specificly.In this study, we constructed the "target+effect" fusion tumor gene therapy system which used the mouse FⅦ light chain (mLFⅦ) as target molecule and Fc as effect molecule. First, in order to study the target function of mLFⅦ, we constructed the tracer plasmid which express red fluorescence protein and yellow fluorescence protein(YFP):pSCIgκ-mLFⅦ-E2, pSCIgK-mLFVH-YFP. Second, we constructed the pSCIgK-mLFⅦ-Fc tumor therapy plasmid which express mLFⅦ and Fc fusion protein. Third, we constructed green fluorescence human liver cancer line SK-Hep-1-G xenografts in nude mouse in order to detect tumor growth situation easily. Forth, We constructed over-expression TF human lung tumor cancer line NCI-H292 xenografts and low-expression TF human lung tumor cancer line A549 xenografts in nude mouse to study wheather the tumor treatment can be influenced by the amount of TF expression.Combination of electrotransfer and Pluronic L64 can be a safe and effective gene delivery method in skeletal muscle, so we use this method to deliver plasmid into skeletal muscle cells of nude mouse xenografts model, with the help of signal peptide the purpose protein which encoded by muscle cells can be secreted into the extracellular, the secreted purpose fusion protein should be transported in the blood to the vasculature of the xenograft where the mLFⅦ domain can interact with the TF targets on the tumor vascular endothelial cells and tumor cells, The Fc domain of the fusion protein should activate an immune response against the targeted tumor vascular endothelial cells and tumor cells by components of the immune system. The results reported here demonstrate that the growth of tumor xenograph, especially expressing a high level of TF can be therapy by this tumor gene therapy system.