新型Ad/AAV嵌合病毒携带全长抗体基因对肿瘤治疗作用的研究
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摘要
实验证明肿瘤的基因靶向治疗可以提高治疗效果。研究人员利用基因治疗的方法,用病毒做为载体将全长抗体基因导入细胞,随着基因的表达,细胞自身产生的抗体就可以杀死肿瘤细胞。在2004年,我们实验室最早构建了携带全长抗体基因的腺病毒,实验证明其对肿瘤有良好的杀伤效果。但由于腺病毒本身的缺点导致抗体基因的表达不长效,进而限制了其临床应用。所以在基因治疗过程中,载体是基因治疗的关键。目前用于基因治疗的病毒载体有:腺相关病毒载体(AAV)、腺病毒载体、逆转录病毒载体以及慢病毒载体等,但各种载体都不尽如人意。
腺相关病毒载体(AAV)基因组两端的两个倒转末端重复序列(ITR)具有可以完成病毒自身复制、包装的能力,并且可以形成AAV环形中间体,这些中间体ITR之间为两个开放阅读框,里面可以插入目的基因。根据这一的特点,我们在腺病毒载体里重组加入两个AAV的ITR,然后在ITR之间插入全长抗体基因序列,病毒感染靶细胞后,抗体基因可以在宿主染色体外形成环形中间体独立表达。这种新型嵌合病毒载体集中了腺病毒容量大、起效快的优点,又具有AAV可以长效表达的优点,是一种新型的理想病毒载体。
本文先是通过Overlap-PCR的方法构建了AAV上游ITR(uITR)和下游ITR (dITR)融合基因片段,再将该融合基因重组进入5型腺病毒中构成新型嵌合病毒载体,然后在新型病毒的ITR中插入抗体Cetuximab(AT2)基因片段构成携带全长抗体的新型嵌合病毒A/V659-AT2。
以实验室已有携带同样抗体基因的重组腺病毒载体AD359-AT2为对照组,检测新型病毒在嵌合了AAV ITR后对抗体基因表达的影响。收集两种病毒感染细胞2、4、6、8天后的细胞上清,通过ELISA和Western blot的方法检测Cetuximab(AT2)抗体的表达情况。ELISA结果发现,这两种病毒在体外都能够顺利表达AT2抗体蛋白,和商品化的Cetuximab大小分子量相同。并且抗体的表达量在前4天的相差不大,但在第6天对照组的抗体表达量开始下降,到第8天时表达量由最高时的138.82ng/ml降为58.49 ng/ml;Western Blot显示其在第6天时轻链重链开始不匹配,在第8天时轻链条带不明显。但携带AAV ITR的新型Ad/AAV嵌合病毒的抗体表达量比较恒定,一直在110ng/ml左右,Western Blot显示其轻重链表达量相匹配,说明表达的抗体具有生物活性。
以携带荧光蛋白基因的重组腺病毒载体AD359-EGFP为对照,用新型Ad/AAV嵌合载体A/V659-EGFP感染NK92细胞,在荧光显微镜下观察EGFP的表达情况,发现对照组的GFP蛋白表达情况在第6天时发生减弱,但新型嵌合载体感染的细胞GFP可以长效表达。因此我们可以把携带抗体基因的新型病毒载体和NK细胞联合使用,利用NK对肿瘤细胞的天然靶向性,携带病毒载体在肿瘤细胞周围富集,从而使抗体在肿瘤中大量长效表达,达到杀死肿瘤的目的,为以后的临床应用打下了基础。
It is fund that the cancer targeting therapy is an important strategy to cure the tumor. useing gene therapy, the virus carry the full length antibody genes into cells, the gene expression in cells can last for a long time, the cells own antibodies can kill tumor cells. In 2004, our laboratory constructed the adenovirus carrying full-length antibody gene for the first time and show its good anti-tumor effect. However, due to shortcomings of adenovirus, antibody genes can’t be expressed long enough and limits the further development of clinical applications. In gene therapy there are adeno-associated virus vector (AAV), adenovirus vectors, retroviral vectors and lentiviral vectors , etc., but all of them are not perfect.
Because ITR is the important for AAV copies and packagin as well as formed AAV circular intermediates, these intermediates between the ITR is the open reading frames, which characteristics of the target gene can be inserted. we insert the two AAV ITR in the adenovirus vector and then inserted full-length antibody genes sequences between the ITRs.When viral infect of target cells, antibody genes can form the ring outside the host chromosome and independent expression. This new hybrid adenovirus vector with large capacity , antibody genes also can be expresed for a long-time. It’s a ideal new virus vector.
Upstream AAV ITR (uITR) and downstream ITR (dITR) fusion gene fragment was geted by Overlap-PCR, and then insert the fusion gene into adenovirus vector, and then full-length antibody Cetuximab (AT2) gene fragments was inserted between the ITRs.This new virus is called A/V659-AT2.
Recombinant adenovirus AD359-AT2 carrying the same antibodie can be used as the control group, the Cetuximab (AT2) antibody expressed in cell supernatant was detected by ELISA and Western blot. At the 2,4,6,8 day collecte cell supernatant, ELISA results showed that both viruses are able to successfully expression of Cetuximab (AT2) antibody in vitro, and molecular weight are the same as the commercialization one. In the first 4 days, the expression of antibodies are same, but at the first 6 day,the antibody expression in the control group began to decrease, at the 8 day, the expression of the antiboday was reduced from 138.82ng/ml(2 day) to 58.49 ng / ml ; Western Blot shown at the first 6 day, the light chain does not match to the heavy chain and in the 8th day ,the light chain is not obvious. But with AAV ITR's,the new Ad / AAV hybrid virus’s express the antibodies relatively constant, around 110ng/ml, Western Blot shows the expression of its light and heavy chain are matched . It means the antibodies have biological activity.
Use recombinant adenovirus vector carried fluorescent protein as a control, the new Ad / AAV hybrid vector carried fluorescent protein infect NK92 cells. Then observe EGFP’s expression under a fluorescence microscope.It was found in the control group the expression of GFP is damed at the 6 day, but New vector infected cells can be long-lasting expression of GFP. Therefore, we can use the viral vectors conjunction with NK cells. As NK cells naturaly targeting tumor cells, viral vectors can be carried to tumor cells, so that a large number of antibody can be expressed in tumors for a long time, so as to kill the tumor cells.
腺相关病毒载体(AAV)基因组两端的两个倒转末端重复序列(ITR)具有可以完成病毒自身复制、包装的能力,并且可以形成AAV环形中间体,这些中间体ITR之间为两个开放阅读框,里面可以插入目的基因。根据这一的特点,我们在腺病毒载体里重组加入两个AAV的ITR,然后在ITR之间插入全长抗体基因序列,病毒感染靶细胞后,抗体基因可以在宿主染色体外形成环形中间体独立表达。这种新型嵌合病毒载体集中了腺病毒容量大、起效快的优点,又具有AAV可以长效表达的优点,是一种新型的理想病毒载体。
本文先是通过Overlap-PCR的方法构建了AAV上游ITR(uITR)和下游ITR (dITR)融合基因片段,再将该融合基因重组进入5型腺病毒中构成新型嵌合病毒载体,然后在新型病毒的ITR中插入抗体Cetuximab(AT2)基因片段构成携带全长抗体的新型嵌合病毒A/V659-AT2。
以实验室已有携带同样抗体基因的重组腺病毒载体AD359-AT2为对照组,检测新型病毒在嵌合了AAV ITR后对抗体基因表达的影响。收集两种病毒感染细胞2、4、6、8天后的细胞上清,通过ELISA和Western blot的方法检测Cetuximab(AT2)抗体的表达情况。ELISA结果发现,这两种病毒在体外都能够顺利表达AT2抗体蛋白,和商品化的Cetuximab大小分子量相同。并且抗体的表达量在前4天的相差不大,但在第6天对照组的抗体表达量开始下降,到第8天时表达量由最高时的138.82ng/ml降为58.49 ng/ml;Western Blot显示其在第6天时轻链重链开始不匹配,在第8天时轻链条带不明显。但携带AAV ITR的新型Ad/AAV嵌合病毒的抗体表达量比较恒定,一直在110ng/ml左右,Western Blot显示其轻重链表达量相匹配,说明表达的抗体具有生物活性。
以携带荧光蛋白基因的重组腺病毒载体AD359-EGFP为对照,用新型Ad/AAV嵌合载体A/V659-EGFP感染NK92细胞,在荧光显微镜下观察EGFP的表达情况,发现对照组的GFP蛋白表达情况在第6天时发生减弱,但新型嵌合载体感染的细胞GFP可以长效表达。因此我们可以把携带抗体基因的新型病毒载体和NK细胞联合使用,利用NK对肿瘤细胞的天然靶向性,携带病毒载体在肿瘤细胞周围富集,从而使抗体在肿瘤中大量长效表达,达到杀死肿瘤的目的,为以后的临床应用打下了基础。
It is fund that the cancer targeting therapy is an important strategy to cure the tumor. useing gene therapy, the virus carry the full length antibody genes into cells, the gene expression in cells can last for a long time, the cells own antibodies can kill tumor cells. In 2004, our laboratory constructed the adenovirus carrying full-length antibody gene for the first time and show its good anti-tumor effect. However, due to shortcomings of adenovirus, antibody genes can’t be expressed long enough and limits the further development of clinical applications. In gene therapy there are adeno-associated virus vector (AAV), adenovirus vectors, retroviral vectors and lentiviral vectors , etc., but all of them are not perfect.
Because ITR is the important for AAV copies and packagin as well as formed AAV circular intermediates, these intermediates between the ITR is the open reading frames, which characteristics of the target gene can be inserted. we insert the two AAV ITR in the adenovirus vector and then inserted full-length antibody genes sequences between the ITRs.When viral infect of target cells, antibody genes can form the ring outside the host chromosome and independent expression. This new hybrid adenovirus vector with large capacity , antibody genes also can be expresed for a long-time. It’s a ideal new virus vector.
Upstream AAV ITR (uITR) and downstream ITR (dITR) fusion gene fragment was geted by Overlap-PCR, and then insert the fusion gene into adenovirus vector, and then full-length antibody Cetuximab (AT2) gene fragments was inserted between the ITRs.This new virus is called A/V659-AT2.
Recombinant adenovirus AD359-AT2 carrying the same antibodie can be used as the control group, the Cetuximab (AT2) antibody expressed in cell supernatant was detected by ELISA and Western blot. At the 2,4,6,8 day collecte cell supernatant, ELISA results showed that both viruses are able to successfully expression of Cetuximab (AT2) antibody in vitro, and molecular weight are the same as the commercialization one. In the first 4 days, the expression of antibodies are same, but at the first 6 day,the antibody expression in the control group began to decrease, at the 8 day, the expression of the antiboday was reduced from 138.82ng/ml(2 day) to 58.49 ng / ml ; Western Blot shown at the first 6 day, the light chain does not match to the heavy chain and in the 8th day ,the light chain is not obvious. But with AAV ITR's,the new Ad / AAV hybrid virus’s express the antibodies relatively constant, around 110ng/ml, Western Blot shows the expression of its light and heavy chain are matched . It means the antibodies have biological activity.
Use recombinant adenovirus vector carried fluorescent protein as a control, the new Ad / AAV hybrid vector carried fluorescent protein infect NK92 cells. Then observe EGFP’s expression under a fluorescence microscope.It was found in the control group the expression of GFP is damed at the 6 day, but New vector infected cells can be long-lasting expression of GFP. Therefore, we can use the viral vectors conjunction with NK cells. As NK cells naturaly targeting tumor cells, viral vectors can be carried to tumor cells, so that a large number of antibody can be expressed in tumors for a long time, so as to kill the tumor cells.
引文
1. Sakurai, F., et al., Characterization of in vitro and in vivo gene transfer properties of adenovirus serotype 35 vector. Mol Ther, 2003. 8(5): p. 813-21.
2. Kotin, R., et al., Site-specific integration by adeno-associated virus. Proceedings of the National Academy of Sciences of the United States of America, 1990. 87(6): p. 2211.
3. Haberman, R., T. McCown, and R. Samulski, Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element. Journal of virology, 2000. 74(18): p. 8732.
4. Musatov, S., et al., A cis-acting element that directs circular adeno-associated virus replication and packaging. J Virol, 2002. 76(24): p. 12792-802.
5.曹佐武, et al., Itr缺陷对aav病毒包装与感染力的影响.生物化学与生物物理进展, 2008. 35(2): p. 224-230.
6.张凤兰,文朝阳, and丁卫,腺相关病毒基因治疗载体的改良与应用.首都医科大学学报, 2009. 30(004): p. 565-573.
7. Fisher, K., et al., A novel adenovirus-adeno-associated virus hybrid vector that displays efficient rescue and delivery of the AAV genome. Human gene therapy, 1996. 7(17): p. 2079-2087.
8. Jacoby, D., C. Fraefel, and X. Breakefield, Hybrid vectors: a new generation of virus-based vectors designed to control the cellular fate of delivered genes. Gene therapy, 1997. 4(12): p. 1281.
9.舒静波, et al.,腺病毒-腺相关病毒嵌合载体的研发趋势.华侨大学学报:自然科学版, 2008. 29(2): p. 172-176.
10. Shayakhmetov, D., et al., A high-capacity, capsid-modified hybrid adenovirus/adeno-associated virus vector for stable transduction of human hematopoietic cells. Journal of virology, 2002. 76(3): p. 1135.
11.徐学武, et al.,表达人内啡肽的腺病毒/腺相关杂合病毒的构建及体外表达分析.微生物学报, 2005. 45(6): p. 856-859.
12. Recchia, A., et al., Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector. Proceedings of the National Academy of Sciences, 1999. 96(6): p. 2615.
13. Goncalves, M., et al., Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability. The Journal of Virology, 2005. 79(5): p. 3146.
14. Gon alves, M., et al., Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system. PLoS One, 2008. 3(8): p. 3084.
15. Weiner, L. An overview of monoclonal antibody therapy of cancer. 1999: Semin Oncol.
16. Weiner, L., M. Dhodapkar, and S. Ferrone, Monoclonal antibodies for cancer immunotherapy. The Lancet, 2009. 373(9668): p. 1033-1040.
17.马力文,靶向治疗药物临床应用进展.中国微创外科杂志, 2005. 5(11): p. 962-964.
18. Cunningham, D., et al., Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. New England Journal of Medicine, 2004. 351(4): p. 337.
19. Bonner, J., et al. Cetuximab prolongs survival in patients with locoregionally advanced squamous cell carcinoma of head and neck: a phase III study of high dose radiation therapy with or without cetuximab. 2004.
20. Bourhis, J., et al., Phase I/II study of cetuximab in combination with cisplatin or carboplatin and fluorouracil in patients with recurrent or metastatic squamous cell carcinoma of the head and neck. Journal of Clinical Oncology, 2006. 24(18): p. 2866.
21. Pfister, D., et al., Concurrent cetuximab, cisplatin, and concomitant boost radiotherapy for locoregionally advanced, squamous cell head and neck cancer: a pilot phase II study of a new combined-modality paradigm. Journal of Clinical Oncology, 2006. 24(7): p. 1072.
22. Kelly, K., et al. A multi-centered phase I/II study of cetuximab in combination with paclitaxel and carboplatin in untreated patients with stage IV non-small cell lung cancer. 2003.
23. Chan, A., et al., Multicenter, phase II study of cetuximab in combination with carboplatin in patients with recurrent or metastatic nasopharyngeal carcinoma. Journal of Clinical Oncology, 2005. 23(15): p. 3568.
24. Wang, X., S. Ponnazhagan, and A. Srivastava, Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions. Journal of virology, 1996. 70(3): p. 1668.
25. GONG, J., G. Maki, and H. Klingemann, Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia, 1994. 8(4): p. 652-658.
26. Maki, G., Ex vivo purging of stem cell autografts using cytotoxic cells. Journal of Hematotherapy & Stem Cell Research, 2001. 10(4): p. 545-551.
2. Kotin, R., et al., Site-specific integration by adeno-associated virus. Proceedings of the National Academy of Sciences of the United States of America, 1990. 87(6): p. 2211.
3. Haberman, R., T. McCown, and R. Samulski, Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element. Journal of virology, 2000. 74(18): p. 8732.
4. Musatov, S., et al., A cis-acting element that directs circular adeno-associated virus replication and packaging. J Virol, 2002. 76(24): p. 12792-802.
5.曹佐武, et al., Itr缺陷对aav病毒包装与感染力的影响.生物化学与生物物理进展, 2008. 35(2): p. 224-230.
6.张凤兰,文朝阳, and丁卫,腺相关病毒基因治疗载体的改良与应用.首都医科大学学报, 2009. 30(004): p. 565-573.
7. Fisher, K., et al., A novel adenovirus-adeno-associated virus hybrid vector that displays efficient rescue and delivery of the AAV genome. Human gene therapy, 1996. 7(17): p. 2079-2087.
8. Jacoby, D., C. Fraefel, and X. Breakefield, Hybrid vectors: a new generation of virus-based vectors designed to control the cellular fate of delivered genes. Gene therapy, 1997. 4(12): p. 1281.
9.舒静波, et al.,腺病毒-腺相关病毒嵌合载体的研发趋势.华侨大学学报:自然科学版, 2008. 29(2): p. 172-176.
10. Shayakhmetov, D., et al., A high-capacity, capsid-modified hybrid adenovirus/adeno-associated virus vector for stable transduction of human hematopoietic cells. Journal of virology, 2002. 76(3): p. 1135.
11.徐学武, et al.,表达人内啡肽的腺病毒/腺相关杂合病毒的构建及体外表达分析.微生物学报, 2005. 45(6): p. 856-859.
12. Recchia, A., et al., Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector. Proceedings of the National Academy of Sciences, 1999. 96(6): p. 2615.
13. Goncalves, M., et al., Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability. The Journal of Virology, 2005. 79(5): p. 3146.
14. Gon alves, M., et al., Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system. PLoS One, 2008. 3(8): p. 3084.
15. Weiner, L. An overview of monoclonal antibody therapy of cancer. 1999: Semin Oncol.
16. Weiner, L., M. Dhodapkar, and S. Ferrone, Monoclonal antibodies for cancer immunotherapy. The Lancet, 2009. 373(9668): p. 1033-1040.
17.马力文,靶向治疗药物临床应用进展.中国微创外科杂志, 2005. 5(11): p. 962-964.
18. Cunningham, D., et al., Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. New England Journal of Medicine, 2004. 351(4): p. 337.
19. Bonner, J., et al. Cetuximab prolongs survival in patients with locoregionally advanced squamous cell carcinoma of head and neck: a phase III study of high dose radiation therapy with or without cetuximab. 2004.
20. Bourhis, J., et al., Phase I/II study of cetuximab in combination with cisplatin or carboplatin and fluorouracil in patients with recurrent or metastatic squamous cell carcinoma of the head and neck. Journal of Clinical Oncology, 2006. 24(18): p. 2866.
21. Pfister, D., et al., Concurrent cetuximab, cisplatin, and concomitant boost radiotherapy for locoregionally advanced, squamous cell head and neck cancer: a pilot phase II study of a new combined-modality paradigm. Journal of Clinical Oncology, 2006. 24(7): p. 1072.
22. Kelly, K., et al. A multi-centered phase I/II study of cetuximab in combination with paclitaxel and carboplatin in untreated patients with stage IV non-small cell lung cancer. 2003.
23. Chan, A., et al., Multicenter, phase II study of cetuximab in combination with carboplatin in patients with recurrent or metastatic nasopharyngeal carcinoma. Journal of Clinical Oncology, 2005. 23(15): p. 3568.
24. Wang, X., S. Ponnazhagan, and A. Srivastava, Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions. Journal of virology, 1996. 70(3): p. 1668.
25. GONG, J., G. Maki, and H. Klingemann, Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia, 1994. 8(4): p. 652-658.
26. Maki, G., Ex vivo purging of stem cell autografts using cytotoxic cells. Journal of Hematotherapy & Stem Cell Research, 2001. 10(4): p. 545-551.