An Experimental Study Of Preparation For Controllable Live Tumor Cellular Vaccine Of Rat Glioma

Gliomas are the most common primary tumors of the central nervous system (CNS). About 40～60% patients'diagnosis of brain tumor are gliomas. The prognosis for most patients harboring gliomas remains grim despite advances in surgery, radiation therapy, and chemotherapy. The mean survival time of maliganant glioma patients, such as glioblastoma, is still about 12～14 months. However, recent decades, the advances of immunobiology and molecular biological knowledge about CNS and gliomas make the promising hope for glioma immnotherapies seem increasingly feasible. Amone them, the therapeutic tumor vaccine is a hot field of research for glioma immunotherapy.As reported for other types of cancers, glioma-induced immunosuppressoin has a number of known pathways, such as weak immunogenicity, low level expression of MHC molecules, lack of costimulatory molecules and adhesion molecules, secreation of immunosuppression cytokines. All above mechanisms induce immunosuppresion status in tumor local site and the body immune system. Many glioma immunothery approaches have been proposed, including administering nonspecific immune adjuvants (cytokines), antitumor antibodies (serotherapy), antitumor lymphocytes (adoptive immunotherapy), and antitumor vaccines (active immunotherapy).Several cytokines have been administered systemically in glioma immunotherap-eutic studies, including IL-2, IL-4, IL-12, IFNα, INFγand TNFα. Interleukin-12 is a heterodimeric protein consisting of two subunits (p35 and p40) and is secreted by antigen-presenting cells such as dendritic cells and macrophages. It enhances the proliferation and cytolytic functions of T cells and NK cells, and promotes these effector cells to produce a number of other cytokines, including IFNr. Furthermore, IL-12 directs the differentiation of uncommitted T cells toward T helper type 1 cells, which is critical for cell-mediated antitumor immunity. IL-12 also has been shown to have po- tent antiangiogeneic activity. It can also enhance the expression levels of MHC mole- cules and adhesion molecules. IL-12 is one of the most efficient antitumor cytokines. It is a bridge linking innate and acquired immunity.In preclinical and clinical trials, cytokines therapeutic activity appears to require high doses that result in treatment-limiting cerebral edema. However, immunogene therapy can overcome this obstacle. Many cancer immunogene therapy studies have focused on genetically engineered tumor cell vaccines. The persistent expressoin of immunogenes in tumor local sites is able to elicit especific antitumor immune responses. Usually, genetically modified tumor cells are attenuated by irradiation to prevent further cell division and proliferation. This is an important factor of lower effic- ience of a dead vaccine than a live. Several reports demonstrated that the secretory volume of cytokines declined dramatically when the immunogene modified tumor cell vaccines were killed by irradiation.Meanwhile, most gene therapy clinical trials for malignant gliomas have been based on the delivery of the suicide gene, herpes simplex viral thymidine kinase (HSV- TK), followed by ganciclovir (GCV) administration. The HSV-TK enzyme phosphorylates the prodrug GCV into the active compound GCV triphosphate, an inhibitor of DNA synthesis that leads to cell death. A useful feature of HSV-TK/GCV gene therapy is the 'bystander effect', that is, killing of nontransduced neighbor cells by GCV treatment. This effect is mediated by transfer of toxic metabolites and by stimu- lation of an immune response involving T cells and natural killer (NK) cells.Taking advantage of HSV-TK/GCV system as a "death swtich" to contral a "live" cytokine (rIL-12) and TK genes modified rat glioma celluar vaccine proliferation in vitro and in vivo; this creative and novel strategy of glioma vaccine research item was supported by a Natural Science Fund of GD Province (001122) and a Medical Fund of PLA (01MA038) in 2000.First, a eukaryotic expression vector containing rat interleukin-12 was construted. Interleukin-12 is a heterodimeric protein consisting of p35 and p40 two subunits. The rat interleukin-12 p35 and p40 cDNA fragments were obtained from the total RNA of Wistar rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) by means of reverse transcription and polymerase chain reaction (RT-PCR) respectively. Applying molecular cloning techniques, The amplified and purified fragments were inserted into pcDNA3.1(-)/myc-his A, an eukaryotic expression plasmid, by two steps. Both p40 and p35 cDNAs were linked by internal ribosom entry site (IRES). The constructed plasmind pcDNA3.1/p40-IRES-p35 (pcDNA3.1/rIL-12) was identified by PCR method, digested with restriction enzymes and DNA sequence analysis. The results of DNA sequence analysis demonstrated that the sequences of p40 and p35 cDNA were identical to those of NM 022611 and NM 053390 in GenBank respectively. The electrophoresis strips of constructed plasmid by means of PCR and digested with restriction enzymes were coincident with expectation.Second, to estiblish a monoclonal cellular strain of rat glioma cell 9L/rIL-12. The wild type rat glioma 9L cells were transfected with the constructed pcDNA3.1/rIL-12 plasmid by Lipofectamine^TM000 or electroperation. Then the transfected cells were cultured with DMEM containing 10% fetal bovine serum, 100 units/ml penicilin G and 100 units/ml streptomycin at 37℃in a humidified 5% CO₂ incubator, and were selected with 600μg/ml G418 for 10～14 days. When the cell clones observed, they were picked up with celluar scraping knife respectively for monoclonal cell strains, and were transferred to a new culture well plate for proliferation and passage. The transducted cells were alse selected by limiting dilution method for monoclonal strains. Obtained 9L/rIL-12 monoclonal cell strains were cultured and surpernatant were collected, and tested for rIL-12 p70 content using an ELISA kit. The strains cells were abstracted for RNA to identify the expression of p35 and p40 genes by RT-PCR. Two strains of 9L/rIL-12, clone-2 and clone-7, were found to secrete rIL-12 p70 protein 1390 and 1621 PG/ 5×10⁶ cells per 72 h. Both p35 and p40 genes were detected in the two strain cells.Three, to construct a lentiviral vector plasmid containing herpes simplex viral thymidine kinase (HSV-TK) gene, and to estiblish the lentiviral expression system of HSV-TK. The fragment of herpes simplex viral thymidine kinase gene was come from pSNAV2.0-TK plasmid digested with EcoR I and Sal I restriction enzymes. The vector was come from pLenti6/5V-EGFP plasmid digested with EcoR I and Xho I restriction enzymes. Then the TK gene was inserted into the vector by conjunction with cleaved termini. The constructed pLenti6/5V-TK plasmid was identified by PCR, restriction enzymes and DNA sequence analysis. After these identifiction, the pLenti6/5V-TK plasmid and the ViraPower^TMPackaging Mix (three packaging plasmids containing pLP1, pLP2 and pLP/VSVG) were cotransfected into 392T cells to produce a replication-incompetent lentivirus. The lentivirus was added to rat glioma 9L cells and selected with Blasticidin (BSD) for the viral titer. The result of pLenti6/5V-TK plasmid DNA sequences analysis demonstrated that the TK sequence was same as GenBank V00470. The electrophoresis strips of constructed plasmid by means of PCR and restriction enzymes were coincident with expectation. The titer of lentivirus obtained from 293T cell surpematant 48 h after cotransfection was 2.7×10⁵TU/ml.Four, to estiblish rIL-12 and TK genes co-modified rat glioma cell vaccine, and to test the ability of HSV-TK/GCV system to contral the live tumor cell vaccine in vitro. Adding the lentivirus containing HSV-TK gene to the 9L/rIL-12 clone-7 cells, and selected with BSD for stably transduced 9L/rIL-12-TK cells. After then, the monoclonal strains were picked. All monoclonal strains were reselected by GCV with a range of concentrations, and picked for those sensitive strains. After reselection, the several passages cells were identified the ability of rIL-12 secretion and TK gene expression in RNA. The strain of 9L/rIL-12-TK clone-29 was found to be satisfact results through all above selections, and was going to act as a live glioma cell vaccine. Five serial passages cells of 9L/rIL-12-TK clone-29 were tested with GCV by MTT method. In vitro, all cells were almost completely killed with 10μg/ml GCV for 96 h. The results of ELISA showed that the tumor cells stably secreted rIL-12 not more affected by integrated HSV-TK gene. The results of MTT proved that HSV-TK/GCV system was able to completely contral the proliferation of the live tumor cell vaccine in vitro.In a conclusion, we were successful to construct a eukaryotic expression vector plasmid containing rIL-12 and a lentiviral vector plasmid containing HSV-TK, and to estiblish the expression system of HSV-TK with lentivirus. Through two steps of gene transduction, 9L/rIL-12-TK, the rat glioma celluar vaccine with IL-12 and TK genes co-modified and stably expressing was obtained. In vitro studies, we proved that HSV-TK/GCV system as a "death swtich" had the complete ability to contral the proliferation of vaccinal tumor cells. Although, the related work in vivo of the live tumor vaccine are going on, a promising hope is worthy to expectation. Meanwhile, the lentivirus as a new gene delivery vehicle will be further explorated in our glimoa immunogene therapy and tumor vaccine studies.