Development Of Tumor Vaccine Of EL-4 Cells Modified By GM-CSF Gene And B7.1 Gene

At present, the majority of leukemia/lymphoma patients can achieve completeremission after receiving powerful combined treatment of chemotherapy and /orradiotherapy and hematopoietic stem cell transplantation. But minimal residualleukemia/lymphoma cells are hard to eradicate thoroughly by current theraputicregimens and would be the root of relapse. Immunogene therapy is thought to be anideal method for eradicating minimal residual desease through enhancement of hostimmunity, elicitation of specific immune response and is expected to improvetreatment effect of tumor and prevent relapse.We have previously shown that the EL-4 cells transfected with B7.1or/andGM-CSF gene can effectively stimulate immune response to lymphoma in vivo. Thetwo genes have synergetic tendency to induce anti-tumor immunity. It might have apotential value of clinical application. In this thesis we plan further to constructco-expression of retroviral vector, prepare tumor vaccine co-expressed double geneand improve the expression of CD80 and secrete GM-CSF in EL-4 cells transfectedby GM-CSF and B7.1 fusagene and are expected to achieve better effect oferadicating minimal residual disease.The subjects of the thesis were focused on the following aspects: 561.Construction of retroviral vector co-expressing mouse GM-CSF and B7.1genes and preparation of retrovirus of pLXSN, pLXSNmGM-CSF, pLXSNmB7.1,pLXSNmGM-B7.1 and these genes modified EL-4 cells. Mouse B7.1 cDNA wascloned by RT-PCR from mouse spleen and was ligated into the retroviral plasmidpLXSN. Mouse GM-CSF and B7.1 cDNA were spliced through a linker sequence byover-lap extention PCR and the mGM-B7.1 fusion gene was also ligated into theretrovirus plasmid pLXSN and its sequence was analyzed. The packaging cell linePA317 was transfected with the plasmids of pLXSN, pLXSNmGM-CSF,pLXSNmB7.1 and pLXSNmGM-B7.1, then G418-resistant clones were obtained inthe selection system containing G418. The retrovirus titer of the supernatants fromthe resistant clones was measured with NIH3T3 cell line. EL-4/pLXSN, EL-4/mGM,EL-4/mB7.1 and EL-4/mGM-B7.1 cells were generated by infecting Wild-type EL-4cells with viral supernatants through 4-6 rounds of transduction, and the expressionsof CD80 and GM-CSF in these cells were detected by flow cytometry andenzyme-linked immunosorbent assay (ELISA).2.The in vivo experiment of induction of anti-lymphoma immunity in C57BL/6mice.(1). Prevention of lymphoma with EL-4/GM-B7.1. C57BL/6 mice were inoculatedwith different times(one to three) tumor vaccines, and then the mice weresubsequently challenged with 5×104 wild-type EL-4 cells. The number of free-tumormice was counted.(2). The therapeutic tests of lymphoma with EL-4/GM-B7.1. C57BL/6 mice wereinoculated with live 5×104 or 1×105 wild-type EL-4 cells. One or five days later, 7they were immunized with 1 × 106 mitomycin-treated EL-4/GM,EL-4/B7.1,EL-4/GM-B7.1, EL-4/pLXSN and EL-4 cells. The survival time wascompared. (3). The pathological and immunohistochemistry examinations. Some of the tumortissues from the experimental mice treated with 6 doses tumor vaccines were stainedwith hematoxylin and eosin for pathological analysis. 3. Construction of eukaryotic expression vector containing human GM-CSF andB7.1 fusion gene from a single cistron. Human GM-CSF cDNA was made pointmutation by megaprimer PCR. Human GM-CSF and B7.1 cDNA were splicedthrough a linker sequence by over-lap extention PCR. The fusion gene was insertedinto pcDNA3 eukaryotic expressing plasmid and its sequence was analyzed. Our major results are as follows: 1. The sequence of mouse B7.1 gene cDNA cloned in pLXSN plasmid was correct.the splicing order, the direction and the sequence in mGM-B7.1 fusion gene insertedinto pLXSN plasmid were all cor...