Construction Of IL-12 Eukaryotic Expression Plasmid And The Study On Anti-Lung Cancer Immunifaction Of Its Tumor Vaccine

Objective: To construct the recombinant plasmid pcDNA3.1(+)- mIL-12 that can express murine IL-12 gene stably in eukaryotic cell, and establish the murine Lewis Lung Carcinoma cel(lLLC)vaccine LLC/ IL-12 which can express murine IL-12 gene stably through recombinant plasmid, then assess the antitumor efficacy of recombinant plasmid and murine LLC cell vaccine which was transfected murine IL-12 gene in an immuno- competent murine Lewis lung cancer model that paralled more closely the clinical therapy of lung cancer, assess the radiosensitive efficacy of murine LLC cell vaccine on murine Lewis lung cancer model. To oberserv the biodistribution of anti-lung cancer monoclonal antibody 1E2 labeled with iodine-131(131I) in mice bearing Lewis lung cancer, evaluate the growth inhibition effect of 1E2 antibody labeled with iodine-131 (131I-1E2 )on mice bearing Lewis lung carcinoma. To observe the synergy effects of IL-12 vaccine on 131I-1E2 antibody targeted therapy.Methods:PORF- mIL-12(Elasti) plasmid was amplified through PCR, the objective segment (mIL-12)was inserted into eukaryotic expression vector pcDNA3.1(+) after enzyme digestion with restriction enzyme HindⅢand EcoRⅠ, mIL-12 was drived by mCMV promoter in pcDNA3.1(+) and transcribed into the same mRNA , the recombinant plasmid was transfected into LLC cell line with lipofectin after identifying it and then measured its expression, cells cycle and apoptosis were observed by FCM. Positive clone cells which can express mIL-12 gene stably were got after selected by G418, cultured them largely after the activity of LLC/mIL-12 was examined. Tumors were established on the right hind leg of C57BL/6 mice with 2×106 LLC cell injected subcutaneously and all mice were divided into 4 group(sn=10)randomly when the tumors reached 0.5-1.0 cm in diameter, the mice were treated by intratumor injection pcDNA3.1(+) -mIL-12 plasmid or LLC/ mIL-12 vaccine on days 1, 4 and 7 respectively, tumor size was measured before and after treatment. Tumor growth curve, CTL and NK activity of spleen cell and tumor lymphocyte infiltration were observed after all mice were killed on day 14. When the study of radiosensitive efficacy was undertaked, all mice were divided into 4 groups(n=10)randomly when the tumors reached 0.5-0.7 cm in diameter, the mices in vaccine and combination group were treated with intratumor injection LLC/ mIL-12 vaccine on days 0, 7 and 14 respectively, and the mice in radiotherapy and combination group were treated with 2Gy local irradiation on days 1, 3 and 5 respectively, but the control group were untreated. All mices were sacrificed on the day 21, tumors size were measured before and after treatment. Tumor's volume, cytotoxic T- lymphocyte (CTL) and natural killer (NK) activity of spleen cell, tumor cell apoptosis were observed after all mice were killed. Tumor tissue of untreated mice bearing Lewis lung cancer were taked out, the expression of antigenic which was called carbamoyl phosphate synthetase (CPS-1) correspanding to 1E2 on Lewis lung carcinoma cell membrane was detected by immunohistochemistry, 1E2 monoclonal antibody was radioiodinated with 131I by chloramine T method, the labelling ratio, radiochemical purity and radioactivity specific activity of labelling antibody were detected, the biodistribution of 131I-1E2 labelling antibody in mice at different timing were observed by tail intravenous injection 131I -1E2 18.5 MBq when tumor models were established. All mice were divided into 4 groups(n=10) randomly when the tumor reached 0.5-0.7 cm in diameter, physiological saline 0.1 m(lBlank control), 1E2 3μg(Positive control), 131I-IgG 18.5 MBq(Negative control), 131I-1E2 18.5 MBq ( Experemental group ) intratumoral injection on days 0, 7 and 14 respectively, tumor sizes and volume were measured twice a week after treatment, and tumor inhibitory rate were calculated. The biopsy change of tumors was observed by electron microscope when mice were executed on the day 21. To observe the synergy effects of IL-12 vaccine on 131I-1E2 antibody targeted therapy, the mIL-12 plasmids were transfected into LLC cells, Na131I was used to label 1E2 Mab, tumors were established on the right hind leg of C57BL/6 mice with 2×106 LLC cell injected subcutaneously, all mice were divided into 4 groups(n=10)randomly when the tumor reached 0.5-1.0 cm in diameter, treated with PBS(intratumoral injected PBS 0.1 ml), GT(intratumoral injected LLC/ mIL-12 vaccine 2×105), RIT ( intratumoral injected 131I -1E2 18.5 MBq ) and GT+RIT(intratumoral injected LLC/ mIL-12 vaccine 2×105 and 131I-1E2 18.5 MBq) respectively. Tumor sizes and volume were measured before and after treatment. Tumor growth inhibitory ratio was calculated. CTL and NK assays were done. The biodistribution of 131I-1E2 labelling antibody in mice at different groups were observed by tail intravenous injection 131I-1E2 18.5 MBq and/or intratumoral injection LLC/ mIL-12 vaccine 2×105 when tumor models were established.Results: The mIL-12 was amplified and inserted into eukaryotic expression vector pcDNA3.1 (+) after enzyme digestion and interlink, identified pcDNA3.1 (+)-mIL-12 plasmid through PCR, enzyme digestion and analyzing sequence, pcDNA3.1 (+)-mIL-12 plasmid have been constructed successfully, mIL-12 has high expression at mRNA and protein level confirmed by RT-PCR and ELISA results, the recombinant plasmid could promote LLC cell apoptosis revealed by FCM, the cultural supernatant of LLC/mIL-12 cells could induce proliferation of murine spleen cells activated by ConA revealed by T cell proliferation experiment. Tumor growth were inhibited in mice treated with pcDNA3.1(+)-mIL-12 plasmid or LLC/ mIL-12 vaccine, the NK and CTL cell activity were improved by mIL-12, and vaccine group surpass plasmid group in two results, there were abundant CD4+,CD8+ T lymphocyte infiltration in the two groups. Tumors were inhibited in mice treated with radiotherapy or LLC/ mIL-12 vaccine, the NK and CTL activity were augmented by mIL-12, and combination group were stronger; But NK and CTL activity were declined in radiotherapy group. 1E2 corresponding antigen (CPS-1) was mostly expressed on tumor cell plasmalemma, labeling ratio of 131I -1E2 antibody was 67.73%, the radiochemical purity was 95.63%, and the radioactivity concentration of 131I-1E2 was mainly distributed in the tumor tissue. Ross tumor volume in experiment group were 0.946±0.153 cm3, weight were 1.802±0.194 g, and tumor inhibitory rate were 72.0% at posttreatment three weeks, there were statistical differences between therapy group and control groups about grass tumor volume, tumor inhibitory rate, tumor weight (p<0.01), same as biopsy change, but there were no statistical differences between control groups (p>0.05). 131I-1E2 has the ability to effectively target tumor cells, especially combined with IL-12 vaccine. RIT+GT can more effectively up-regulate the expression of IL-l2 gene and inhibit the tumor growth than either GT or RIT. NK and CTL cell activities were improved by mIL-12. IL-12 tumor vaccine could promote 131I-1E2 to target tumor tissiue and decreas its disposition in normal tissue. Conclusion:The pcDNA3.1(+)-mIL-12 eukaryotic expression plasmid was constructed and conformed into LLC cell genome successfully, it can express murine IL-12 gene stable in LLC cell line, LLC /mIL-12 cell line has biological activity. Antitumor immune response are induced both in pcDNA3.1(+)-mIL-12 plasmid and LLC/ mIL-12 vaccine group, and the latter is stronger than the former. Antitumor immune response was induced both in radiotherapy and LLC/ mIL-12 vaccine group, and the combination group was stronger. The radiosensitive mechanism of vaccine is to induce tumor cell apoptosis, enhance NK and CTL activity. 131I-1E2 antibody injected intratumor has the ability to target tumour cells therefor inhibit the growth of tumor in vivo, 131I-1E2 possess potential clinical application value, and maybe become the new targeted drug on tumor therapy. LLC/ mIL-12 vaccine has the synergy effects on 131I-1E2 antibody targeted therapy.