| It has been well accepted that Anti-tumor immune response is mainly mediated by T lymphocytes and "two signals" are necessary to activate T lymphocytes. The first signal is provided by TCR-CD3 which is engaged with the antigen peptide-MHC complex, and the second, also named the co-stimulatory signal, is provided by the interaction between co-stimulatory molecules on antigen presenting cells and their counter-receptors on T cells. The absence of co-stimulatory signal can result in immune-resistance of T-cell and death of activated T-cell. A number of co-stimulatory molecules have been demonstrated to date including B7, ICAM, HSPs, 4-IBB etc. B7 is the most important one among the family of co-stimulatory molecules. There is none or little expression of co-stimulatory molecules in tumor, that is one of the important reasons that lead to immunological "tumor-escaping" .Bladder cancer is the most common urological malignant disease in China. Although intravesical administration of Bacillus Calmette-Guerin (BCG) has been accepted as the most effective therapy for superficial bladder cancer and carcinoma in situ of the bladder, 25-40% of the patients never responds to BCG therapy. Furthermore, Toxicity associated with the BCG therapy is frequent with occurrence of severe adverse effects in 5% of patients and life-threatening symptoms in 0.5% of patients.We plan to combine the superiority of BCG and co-stimulatory molecules in the immunotherapy of bladder cancer, (1) BCG: increasing immunogen of tumor, attracting immunocytes and augmenting cytokine production from immune cells; (2) B7-2 (CD86) : providing co-stimulatory signal, activatinglymphocyte, NK cell and other immunocyte. Thus our recombinant BCG for anti-tumor immunotherapy could not only enhance the first signal but also provide co-stimulatory molecules, which could lower BCG toxicity due to the reduced BCG dose while at the same time preserving or enhancing BCG activity against tumors. These favourable synergies between BCG and hB7-2 are believed to contribute to the improved efficacy observed in anti-bladder cancer immunotherapy.Our laboratory has pioneered methods to genetically engineer BCG, which allows it to express biologically active molecules of interest. Some research suggests that the extramembrane part of B7-2 without the transmembrane and intracellular domain possessed biological activity. According to the above, in an attempt to obtain an improved remedy for immunotherapy in bladder cancer, in this study we intended to make a new strain of BCG that was engineered to secrete recombinant human B7-2(IgC+IgV), extracellar domain of B7-2(CD86).Using our established cytokine induction bioassays, we evaluated the newly constructed rBCG-B7-2(IgC+IgV). We demonstrated that rBCG-B7-2(IgC+IgV) possesses enhanced immunogenicity. Compared with wild-type BCG, rBCG significantly enhanced the effects on bladder cancer cells.1. The construction of pYL-hB7-2(IgC+IgV) plasmidMethods The inserted sequence was derived from polymerase chain reaction (PCR) amplification of plasmid including human B7-2 using a pair of primers for the mature form of the cytokine. The upstream primer contained BamHI restriction site and the start codon ATG, whereas the downstream primer contained the stop codon TAG and Xhol restriction site. The pYL-hB7-2 plasmid was constructed by ligating the BamH I- Xho I-digested cDNA inserts for humanB7-2 into the pYL plasmid digested with the same enzymes. E.coli wastransformed with the constructed pYL-hB7-2(IgC+IgV) plasmid according to the manufacture's instruction and selected on kanamycin (30ug/ml) LB agar plate. The structure of E.coli-derived plasmid was verified by restriction analysis using double digestion of BamHI and Xhol, PCR and sequencing using the sense primer 5'-ATCTTACCCATACGACGTCC-3' and antisense primer 5'-GTTAACTACGTCGACATCG-3', both were part of the pYL plasmid sequence.Results The hB7-2(CD86) gene was inserted into the pYL plasmid exactly. The specific expression vector for hB7-2(CD86) gene was construc...
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