免疫增效型广谱抗肿瘤血管生成基因疫苗的基础研究
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摘要
背景与目的：
上世纪70年代由Judah Folkman提出的有关肿瘤血管生成的理论已被广泛接受,目前,抗肿瘤血管生成已经成为肿瘤学基础研究及临床治疗领域中最有前景的策略之一。大量研究证据表明,在众多的血管生成刺激因子中,内皮生长因子(VEGF)及其受体VEGFR2(鼠中称为flk-1,人中称为KDR)对于与肿瘤生长及转移密切相关的血管生成是最重要的,许多旨在通过打破VEGF/VEGFR2传导通路抑制肿瘤血管生成的主动免疫实验研究都取得了比较理想的结果,还有研究表明,与全长基因疫苗相比,编码VEGFR2胞外1-4区的基因疫苗同样能够有效降低小鼠血清中VEGF水平,从而抑制肿瘤的生长及转移。
构建一种能够诱导有效免疫反应基因疫苗的关键是打破对靶抗原的自身免疫耐受,在这项研究中我们主要采取三项措施解决这一问题。首先,选取异种同源的小鼠VEGFR2胞外1-4 IgG样区域(简称mVEGFR2(1-4))作为疫苗的靶抗原；其次,将人白细胞介素12(hIL-12)融合基因插入到载体IRES序列的下游,构建一个下游可以同时表达hIL-12双亚基的双顺反子真核表达载体pVAX1-IRES-hIL12,hIL-12作为对体内细胞和体液免疫系统作用最强的免疫活性因子及一种抗血管生成因子,在基因疫苗中以免疫佐剂的形式发挥作用；最后,为了增强本疫苗的免疫粘附及抗原递呈功能,本研究利用真核表达载体pCI-Fc-GPI,实现了人Igk前导信号肽、人IgG-Fc段及GPI与靶抗原mVEGFR2(1-4)的共同表达,成功构建了免疫增效型广谱抗肿瘤血管生成基因疫苗pVAX1-sig-mVEGFR2(1-4)-Fc-GPI-IRES-hIL 12(简称pVAX1-mVEGFR2-hIL12)。
方法：
通过搭桥PCR获得人白细胞介素12 P35及P40双亚基的融合基因P35-F2A-P40,插入已构建好的DNA疫苗载体pVAX1-IRES下游,构建质粒载体pVAX1-IRES-hIL12;通过RT-PCR的方法从Balb/c小鼠胚胎组织中特异性扩增mVEGFR2(1-4)基因,连接到真核表达载体pVAX1-IRES-hIL12的上游,构建基因疫苗pVAX1-mVEGFR2-hIL12。脂质体法瞬时转染293T细胞,用ELISA、免疫荧光和流式细胞术分别检测293T细胞中hIL-12和mVEGFR2(1-4)基因的蛋白表达。
结果：
特异性扩增得到大小与mVEGFR2(1-4)基因相符的基因片段,序列测定证实与GenBank(GI:57923)上登录的序列一致；酶切鉴定和序列分析表明P35及P40双亚基融合基因P35-F2A-P40与设计完全一致,融合基因在体外细胞培养液检测中获得分泌表达；成功构建了广谱抗肿瘤血管生成基因疫苗pVAX1-mVEGFR2-hIL12。FACS及IMF检测证实重组基因疫苗得到有效表达。
结论：
成功构建了免疫增效型广谱抗肿瘤血管基因疫苗pVAX1-mVEGFR2-hIL12,为后续研究中进一步评价该疫苗抗肿瘤血管生成的效果奠定了必要基础,并为肿瘤免疫治疗领域提供了一种新的疫苗研究方向。
Background and Objective:
The theory of tumor angiogenesis first presented by Dr Judah Folkman in 1970s has been accepted widely, and today anti-angiogenesis has become one of the most exciting and visible areas in cancer research and therapeutics of clinical oncology. Among all of the factors stimulating angiogenesis, compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptor VEGFR2 (also called fetal liver kinse-1[Flk-1] in mice, kinase-containing domain receptor [KDR] in humans)are critically important to the angiogenesis associated with tumor growth and metastasis. Many researches of active immunotherapy targeting tumor angiogenesis through interrupting the signal passway of VEGF/VEGFR2 have achieved respiratory results. There are also experiments showing that compared with full length of VEGFR2, the 1-4 of extracellular IgG-like domains are sufficient to decrease serum VEGF level and to inhibit tumor growth and metastasis specially.
The key point of constructing a gene vaccine and inducing effective immunological reactions is how to break self-immunological tolerance to the target antigens. In this study, three major strategies were presented to solve the problem mentioned. First, the xenogeneic homologous gene of 1-4 extracellular IgG-like domains of mice (mVEGFR2(1-4) for abbreviation) was selected as the anti-angiogenesis antigen. Second, human interleukin-12(hIL-12), which is known as the most effective immunostimulator of cellular and humoral immune systems and one of the angiogenic inhibitors, was inserted into the downstream of IRES sequence to construct a bicistronic eucaryotic expression plasmid vector pVAX1-IRES-hIL12 co-expressing the double subunits of hIL12 serving as an immunoadjuvant. Third, to enhance immue adhering and antigen presenting of the vaccine, coexpression of mVEGFR2(1-4) with signal peptide of human IgK, human IgG-Fc and GPI has been achieved by utilizing the eukaryotic expression vector pCI-Fc-GPI. Furthermore, the gene vaccine pVAX1-sig-mVEGFR2 (1-4)-Fc-GPI-IRES-hIL12 (pVAXl-mVEGFR2-hIL12 for abbreviation) was cloned and amplified specifically.
Methods:
The fusion gene P35-F2A-P40 was amplified by the overlap extension PCR, and inserted into the downstream of the eukaryotic expression vector pVAX1-IRES to construct the plasmid vector pVAX1-IRES-hIL12. The mVEGFR2(1-4) gene cloned from BALB/c mouse embryo tissue using RT-PCR technique was inserted into the upstream of the pVAX1-IRES-hIL12 vector to construct the active immune plasmid vaccine pVAXl-mVEGFR2-hIL12. Under the mediation of lipofectamine, the plasmid was transfected into 293T cells. The transcription and expression of the hIL12 gene and mVEGFR2 (1-4) were identified by ELISA, immunofluorescence and flow cytometry assay respectively.
Results:
The target gene mVEGFR2(1-4) was obtained and identical with the sequences of GenBank (GI:57923). The enzyme digestion and sequence analysis showed that the fusion gene P35-F2A-P40 was constructed successfully as designed, and the expression of hIL12 was demonstrated by ELISA. The recombinant universal gene vaccine against tumor angiogenisis pVAX1-mVEGFR2-hIL12 was constructed successfully. The effective expression of this plasmid had been demonstrated by FACS and IMF.
Conclusion:
The recombinant universal gene vaccine against tumor angiogenisis pVAX1-mVEGFR2-hIL12 was constructed successfully. These results have provided necessary basis to evaluate the anti-angiogenic effects on tumors of the vaccine in the following research and showed promise to the research of the vaccine in the tumor immunotherapy.
上世纪70年代由Judah Folkman提出的有关肿瘤血管生成的理论已被广泛接受,目前,抗肿瘤血管生成已经成为肿瘤学基础研究及临床治疗领域中最有前景的策略之一。大量研究证据表明,在众多的血管生成刺激因子中,内皮生长因子(VEGF)及其受体VEGFR2(鼠中称为flk-1,人中称为KDR)对于与肿瘤生长及转移密切相关的血管生成是最重要的,许多旨在通过打破VEGF/VEGFR2传导通路抑制肿瘤血管生成的主动免疫实验研究都取得了比较理想的结果,还有研究表明,与全长基因疫苗相比,编码VEGFR2胞外1-4区的基因疫苗同样能够有效降低小鼠血清中VEGF水平,从而抑制肿瘤的生长及转移。
构建一种能够诱导有效免疫反应基因疫苗的关键是打破对靶抗原的自身免疫耐受,在这项研究中我们主要采取三项措施解决这一问题。首先,选取异种同源的小鼠VEGFR2胞外1-4 IgG样区域(简称mVEGFR2(1-4))作为疫苗的靶抗原；其次,将人白细胞介素12(hIL-12)融合基因插入到载体IRES序列的下游,构建一个下游可以同时表达hIL-12双亚基的双顺反子真核表达载体pVAX1-IRES-hIL12,hIL-12作为对体内细胞和体液免疫系统作用最强的免疫活性因子及一种抗血管生成因子,在基因疫苗中以免疫佐剂的形式发挥作用；最后,为了增强本疫苗的免疫粘附及抗原递呈功能,本研究利用真核表达载体pCI-Fc-GPI,实现了人Igk前导信号肽、人IgG-Fc段及GPI与靶抗原mVEGFR2(1-4)的共同表达,成功构建了免疫增效型广谱抗肿瘤血管生成基因疫苗pVAX1-sig-mVEGFR2(1-4)-Fc-GPI-IRES-hIL 12(简称pVAX1-mVEGFR2-hIL12)。
方法：
通过搭桥PCR获得人白细胞介素12 P35及P40双亚基的融合基因P35-F2A-P40,插入已构建好的DNA疫苗载体pVAX1-IRES下游,构建质粒载体pVAX1-IRES-hIL12;通过RT-PCR的方法从Balb/c小鼠胚胎组织中特异性扩增mVEGFR2(1-4)基因,连接到真核表达载体pVAX1-IRES-hIL12的上游,构建基因疫苗pVAX1-mVEGFR2-hIL12。脂质体法瞬时转染293T细胞,用ELISA、免疫荧光和流式细胞术分别检测293T细胞中hIL-12和mVEGFR2(1-4)基因的蛋白表达。
结果：
特异性扩增得到大小与mVEGFR2(1-4)基因相符的基因片段,序列测定证实与GenBank(GI:57923)上登录的序列一致；酶切鉴定和序列分析表明P35及P40双亚基融合基因P35-F2A-P40与设计完全一致,融合基因在体外细胞培养液检测中获得分泌表达；成功构建了广谱抗肿瘤血管生成基因疫苗pVAX1-mVEGFR2-hIL12。FACS及IMF检测证实重组基因疫苗得到有效表达。
结论：
成功构建了免疫增效型广谱抗肿瘤血管基因疫苗pVAX1-mVEGFR2-hIL12,为后续研究中进一步评价该疫苗抗肿瘤血管生成的效果奠定了必要基础,并为肿瘤免疫治疗领域提供了一种新的疫苗研究方向。
Background and Objective:
The theory of tumor angiogenesis first presented by Dr Judah Folkman in 1970s has been accepted widely, and today anti-angiogenesis has become one of the most exciting and visible areas in cancer research and therapeutics of clinical oncology. Among all of the factors stimulating angiogenesis, compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptor VEGFR2 (also called fetal liver kinse-1[Flk-1] in mice, kinase-containing domain receptor [KDR] in humans)are critically important to the angiogenesis associated with tumor growth and metastasis. Many researches of active immunotherapy targeting tumor angiogenesis through interrupting the signal passway of VEGF/VEGFR2 have achieved respiratory results. There are also experiments showing that compared with full length of VEGFR2, the 1-4 of extracellular IgG-like domains are sufficient to decrease serum VEGF level and to inhibit tumor growth and metastasis specially.
The key point of constructing a gene vaccine and inducing effective immunological reactions is how to break self-immunological tolerance to the target antigens. In this study, three major strategies were presented to solve the problem mentioned. First, the xenogeneic homologous gene of 1-4 extracellular IgG-like domains of mice (mVEGFR2(1-4) for abbreviation) was selected as the anti-angiogenesis antigen. Second, human interleukin-12(hIL-12), which is known as the most effective immunostimulator of cellular and humoral immune systems and one of the angiogenic inhibitors, was inserted into the downstream of IRES sequence to construct a bicistronic eucaryotic expression plasmid vector pVAX1-IRES-hIL12 co-expressing the double subunits of hIL12 serving as an immunoadjuvant. Third, to enhance immue adhering and antigen presenting of the vaccine, coexpression of mVEGFR2(1-4) with signal peptide of human IgK, human IgG-Fc and GPI has been achieved by utilizing the eukaryotic expression vector pCI-Fc-GPI. Furthermore, the gene vaccine pVAX1-sig-mVEGFR2 (1-4)-Fc-GPI-IRES-hIL12 (pVAXl-mVEGFR2-hIL12 for abbreviation) was cloned and amplified specifically.
Methods:
The fusion gene P35-F2A-P40 was amplified by the overlap extension PCR, and inserted into the downstream of the eukaryotic expression vector pVAX1-IRES to construct the plasmid vector pVAX1-IRES-hIL12. The mVEGFR2(1-4) gene cloned from BALB/c mouse embryo tissue using RT-PCR technique was inserted into the upstream of the pVAX1-IRES-hIL12 vector to construct the active immune plasmid vaccine pVAXl-mVEGFR2-hIL12. Under the mediation of lipofectamine, the plasmid was transfected into 293T cells. The transcription and expression of the hIL12 gene and mVEGFR2 (1-4) were identified by ELISA, immunofluorescence and flow cytometry assay respectively.
Results:
The target gene mVEGFR2(1-4) was obtained and identical with the sequences of GenBank (GI:57923). The enzyme digestion and sequence analysis showed that the fusion gene P35-F2A-P40 was constructed successfully as designed, and the expression of hIL12 was demonstrated by ELISA. The recombinant universal gene vaccine against tumor angiogenisis pVAX1-mVEGFR2-hIL12 was constructed successfully. The effective expression of this plasmid had been demonstrated by FACS and IMF.
Conclusion:
The recombinant universal gene vaccine against tumor angiogenisis pVAX1-mVEGFR2-hIL12 was constructed successfully. These results have provided necessary basis to evaluate the anti-angiogenic effects on tumors of the vaccine in the following research and showed promise to the research of the vaccine in the tumor immunotherapy.
引文
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[2]Leclerc C. New approaches in vaccine development[J]. Comp Immunol Microbiol Infect Dis,2003,26(5-6):329-341.
[3]Folkman,J. Tumor angiogenesis:therapeutic implications. N. Engl. J. Med,1971 (285):1182-1186.
[4]Folkman,J. Anti-angiogenesis:new concept for therapy of solid tumors. Ann. Surg, 1972(175):409-416.
[5]Blagosklonny MV. Antiangiogenic therapy and tumor progression. Cancer Cell,2004(5):13-17.
[6]Ferrara N. VEGF and the quest for tumor angiogenesis factors. Nat Rev Cancer, 2002(2):795-803.
[7]Liu JY, Wei YQ, Yang L, et al. Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood, 2003(102):1815-1823.
[8]Jian Dong, Jun Yang, Ming Qing Chen, et al. A comperative study of gene vaccines encoding different extracellular domains of the vascular endothelial growth factor receptor 2 in the mouse model of colon adenocarcinoma CT-26. Cancer Biology & Therapy.2008.7(4):502-509.
[9]Stern A S, Podlaski FJ, Hulmes JD, et al. Purification to homo-geneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells [J]. Pro Natl Acad Sci USA,1990,87 (17):6808-6812.
[10]Michele DelVecchio, Emilio Bajetta, et al. Interleukin-12:Biological Properties and Clinical Application. Clin Cancer Res,2007;13(16):4677-4685.
[11]Sangro B, Melero I, Qian C, et al. Gene therapy of cancer based on interleukin 12, Current Gene Therapy,2005,5(6):573-581.
[12]Chen B, Timiryasova TM, Andres ML, et al. Evaluation of combined vaccinia virus mediated antitumor gene therapy with p53, IL-2, and IL-12 in a glioma model. Cancer Gene Ther.2000; 7(11):1678-1692.
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[1]Napoleone Ferrara. VEGF and the quest for tumour angiogenesis factors. Nature Reviews Cancer,2002,10:795-803.
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[1]Folkman J. Tumor angiogenesis:therapeutic implications. N Engl J Med,1971, 285(21):1182-1186.
[2]Folkman,J. Anti-angiogenesis:new concept for therapy of solid tumors. Ann Surg,1972(175):409-416.
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[23]Jian Dong, Jun Yang, Ming Qing Chen, et al. A comperative study of gene vaccines encoding different extracellular domains of the vascular endothelial growth factor receptor 2 in the mouse model of colon adenocarcinoma CT-26. Cancer Biology & Therapy.2008.7(4):502-509.
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[2]Leclerc C. New approaches in vaccine development[J]. Comp Immunol Microbiol Infect Dis,2003,26(5-6):329-341.
[3]Folkman,J. Tumor angiogenesis:therapeutic implications. N. Engl. J. Med,1971 (285):1182-1186.
[4]Folkman,J. Anti-angiogenesis:new concept for therapy of solid tumors. Ann. Surg, 1972(175):409-416.
[5]Blagosklonny MV. Antiangiogenic therapy and tumor progression. Cancer Cell,2004(5):13-17.
[6]Ferrara N. VEGF and the quest for tumor angiogenesis factors. Nat Rev Cancer, 2002(2):795-803.
[7]Liu JY, Wei YQ, Yang L, et al. Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood, 2003(102):1815-1823.
[8]Jian Dong, Jun Yang, Ming Qing Chen, et al. A comperative study of gene vaccines encoding different extracellular domains of the vascular endothelial growth factor receptor 2 in the mouse model of colon adenocarcinoma CT-26. Cancer Biology & Therapy.2008.7(4):502-509.
[9]Stern A S, Podlaski FJ, Hulmes JD, et al. Purification to homo-geneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells [J]. Pro Natl Acad Sci USA,1990,87 (17):6808-6812.
[10]Michele DelVecchio, Emilio Bajetta, et al. Interleukin-12:Biological Properties and Clinical Application. Clin Cancer Res,2007;13(16):4677-4685.
[11]Sangro B, Melero I, Qian C, et al. Gene therapy of cancer based on interleukin 12, Current Gene Therapy,2005,5(6):573-581.
[12]Chen B, Timiryasova TM, Andres ML, et al. Evaluation of combined vaccinia virus mediated antitumor gene therapy with p53, IL-2, and IL-12 in a glioma model. Cancer Gene Ther.2000; 7(11):1678-1692.
[1]Napoleone Ferrara. VEGF and the quest for tumour angiogenesis factors. Nature Reviews Cancer,2002,10:795-803.
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