靶向ROR1的嵌合抗原受体修饰T细胞的构建及对ROR1阳性肿瘤细胞的杀伤作用
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摘要
目的探讨靶向Ⅰ型受体酪氨酸激酶样孤儿受体(ROR1)的嵌合抗原受体修饰的T细胞(chimeric antigen receptor T cell, CAR-T)治疗ROR1阳性肿瘤细胞的体外杀伤作用。方法设计并合成CAR基因,通过慢病毒载体质粒pWPXLd,构建靶向ROR1的CAR-T核心质粒,采用BamHⅠ/EcoRⅠ双酶切鉴定,应用流式细胞术检测多种实体瘤癌细胞株表面ROR1的表达,并采用流式细胞术、乳酸脱氢酶(LDH)检测试验检测CAR-T对ROR1阳性肿瘤细胞的杀伤作用,ELISA检测试剂盒测定培养细胞上清中γ-干扰素(IFN-γ)含量。结果构建的CAR-T质粒双酶切鉴定有CAR基因插入,流式细胞术检测结果显示CAR-T感染效率约为47.23%。多种肿瘤细胞不同程度表达ROR1。流式细胞术、LDH检测试验结果提示CAR-T能特异性地杀伤ROR1阳性肿瘤细胞。CAR-T针对ROR1阳性靶细胞能释放更多的IFN-γ(P<0.05)。结论成功构建靶向ROR1的CAR-T,并能特异地杀伤ROR1阳性肿瘤细胞,释放大量IFN-γ,为临床应用提供了实验基础。
Objective To test the killing effect of type Ⅰ receptor tyrosine kinase-like orphan receptor(ROR1) chimeric antigen receptor T cell(CAR-T) on several ROR1-expressing tumor cells in vitro. Methods The CAR gene was designed and synthesized by constructing the lentiviral vector plasmid, and BamHⅠ/EcoRⅠ was used to identify the plasmid. The expression levels of ROR1 among a variety of tumor cell lines were compared using flow cytometry(FCM). The killing effect of CAR-T on positive cells was detected by FCM, the LDH assay and ELISA. Results The double enzyme digestion identified CAR gene was successfully constructed to the lentivirus vector plasmid. FCM detection showed that the efficiency of CAR-T infection was about 47.23%. Multiple tumor cells expressed ROR1 in varying degrees. The FCM and the LDH assay indicated that CAR-T specifically killed ROR1-positive tumor cells. On positive target cells, more interferonI-γ(FN-γ) could be released during the CAR-T killing process than control T(P<0.05). Conclusion We successfully constructed ROR1 CAR-T. CAR-T can specifically kill ROR1-positive tumor cells and cause the release of large amounts of IFN-γ, providing an experimental basis for clinical application.
Objective To test the killing effect of type Ⅰ receptor tyrosine kinase-like orphan receptor(ROR1) chimeric antigen receptor T cell(CAR-T) on several ROR1-expressing tumor cells in vitro. Methods The CAR gene was designed and synthesized by constructing the lentiviral vector plasmid, and BamHⅠ/EcoRⅠ was used to identify the plasmid. The expression levels of ROR1 among a variety of tumor cell lines were compared using flow cytometry(FCM). The killing effect of CAR-T on positive cells was detected by FCM, the LDH assay and ELISA. Results The double enzyme digestion identified CAR gene was successfully constructed to the lentivirus vector plasmid. FCM detection showed that the efficiency of CAR-T infection was about 47.23%. Multiple tumor cells expressed ROR1 in varying degrees. The FCM and the LDH assay indicated that CAR-T specifically killed ROR1-positive tumor cells. On positive target cells, more interferonI-γ(FN-γ) could be released during the CAR-T killing process than control T(P<0.05). Conclusion We successfully constructed ROR1 CAR-T. CAR-T can specifically kill ROR1-positive tumor cells and cause the release of large amounts of IFN-γ, providing an experimental basis for clinical application.
引文
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[17] PARK JH, GEYER MB, BRENTJENS RJ. CD19-targeted CAR T-cell therapeutics for hematologic malignancies:interpreting clinical outcomes to date. Blood,2016,127(26):3312-3320.
[18] BERGER C, SOMMERMEYER D, HUDECEK M, et al. Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells. Cancer Immunol Res,2015,3(2):206-216.
[19] JOHNSON LA, MORGAN RA, DUDLEY ME, et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood,2009,114(3):535-546.
[20] JUNG EH, LEE HN, HAN GY, et al. Targeting ROR1 inhibits the self-renewal and invasive ability of glioblastoma stem cells. Cell Biochem Funct,2016,34(3):149-157.
[21] BRENTJENS RJ, RIVIERE I, PARK JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood,2011,118(18):4817-4828.
[22] PORTER DL, LEVINE BL, KALOS M, et al. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med,2011,365(8):725-733.
[23] ZHANG BL, QIN DY, MO ZM, et al. Hurdles of CAR-T cell-based cancer immunotherapy directed against solid tumors. Sci China Life Sci,2016,59(4):340-348.
[24] ZHANG H, YE ZL, YUAN ZG, et al. New Strategies for the treatment of solid tumors with CAR-T cells. Int J Biol Sci,2016,12(6):718-729.
[25] PARK HJ, KUSNADI A, LEE EJ, et al. Tumor-infiltrating regulatory T cells delineated by upregulation of PD-1 and inhibitory receptors. Cell Immunol,2012,278(1/2):76-83.
[26] BROWN CE, ALIZADEH D, STARR R, et al. Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J Med,2016,375(26):2561-2569.
[27] KATZ SC, BURGA RA, MCCORMACK E, et al. Phase I hepatic immunotherapy for metastases study of intra-arterial chimeric antigen receptor-modified T-cell therapy for CEA+ liver metastases. Clin Cancer Res,2015,21(14):3149-3159.
[28] ZHANG H, QIU J, YE C, et al. ROR1 expression correlated with poor clinical outcome in human ovarian cancer. Sci Rep,2014,4:5811[2018-09-16].https://www.nature.com/articles/srep05811. doi:10.1038/srep05811.
[2] SHABANI M, ASGARIAN-OMRAN H, VOSSOUGH P, et al. Expression profile of orphan receptor tyrosine kinase (ROR1) and Wilms’ tumor gene 1 (WT1) in different subsets of B-cell acute lymphoblastic leukemia. Leuk Lymphoma,2008,49(7):1360-1367.
[3] BALAKRISHNAN A, GOODPASTER T, RANDOLPH-HABECKER J, et al. Analysis of ROR1 protein expression in human cancer and normal tissues. Clin Cancer Res,2016,23(12):3061-3071.
[4] BASKAR S, KWONG KY, HOFER T, et al. Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia. Clin Cancer Res,2008,14(2):396-404.
[5] DANESHMANESH AH, PORWIT A, HOJJAT-FARSANGI M, et al. Orphan receptor tyrosine kinases ROR1 and ROR2 in hematological malignancies. Leuk Lymphoma,2013,54(4):843-850.
[6] RABBANI H, OSTADKARAMPOUR M, DANESH MANESH AH, et al. Expression of ROR1 in patients with renal cancer-a potential diagnostic marker. Iran Biomed J,2010,14(3):77-82.
[7] ZHANG S, CHEN L, WANG-RODRIGUEZ J, et al. The onco-embryonic antigen ROR1 is expressed by a variety of human cancers. Am J Pathol,2012,181(6):1903-1910.
[8] ZHANG S, CHEN L, CUI B, et al. ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth. PLoS One,2012, 7(3):e31127[2018-09-16]. https://doi.org/ 10.1371/journal.pone.0031127.
[9] CUI B, ZHANG S, CHEN L, et al. Targeting ROR1 inhibits epithelial-mesenchymal transition and metastasis. Cancer Res,2013,73(12):3649-3660.
[10] HOJJAT-FARSANGI M, GHAEMIMANESH F, DANESHMANESH AH, et al. Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells. PLoS One,2013,8(4):e61167[2018-09-16]. https://doi.org/ 10.1371/journal.pone.0061167.
[11] DANESHMANESH AH, HOJJAT-FARSANGI M, KHAN AS, et al. Monoclonal antibodies against ROR1 induce apoptosis of chronic lymphocytic leukemia (CLL) cells. Leukemia,2012,26(6):1348-1355.
[12] SADELAIN M, BRENTJENS R, RIVIéRE I. The promise and potential pitfalls of chimeric antigen receptors. Curr Opin Immunol,2009,21(2):215-223.
[13] HOMBACH A, HOMBACH AA, ABKEN H. Adoptive immunotherapy with genetically engineered T cells:modification of the IgG1 Fc ‘spacer’ domain in the extracellular moiety of chimeric antigen receptors avoids ‘off-target’ activation and unintended initiation of an innate immune response. Gene Ther,2010,17(10):1206-1213.
[14] HUDECEK M, SOMMERMEYER D, KOSASIH PL, et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res,2015,3(2):125-135.
[15] HARRIS DT, KRANZ DM. Adoptive T cell therapies:a comparison of T cell receptors and chimeric antigen receptors. Trends Pharmacol Sci,2016,37(3):220-230.
[16] SADELAIN M, BRENTJENS R, RIVIERE I. The basic principles of chimeric antigen receptor design. Cancer Discov,2013,3(4):388-398.
[17] PARK JH, GEYER MB, BRENTJENS RJ. CD19-targeted CAR T-cell therapeutics for hematologic malignancies:interpreting clinical outcomes to date. Blood,2016,127(26):3312-3320.
[18] BERGER C, SOMMERMEYER D, HUDECEK M, et al. Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells. Cancer Immunol Res,2015,3(2):206-216.
[19] JOHNSON LA, MORGAN RA, DUDLEY ME, et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood,2009,114(3):535-546.
[20] JUNG EH, LEE HN, HAN GY, et al. Targeting ROR1 inhibits the self-renewal and invasive ability of glioblastoma stem cells. Cell Biochem Funct,2016,34(3):149-157.
[21] BRENTJENS RJ, RIVIERE I, PARK JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood,2011,118(18):4817-4828.
[22] PORTER DL, LEVINE BL, KALOS M, et al. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med,2011,365(8):725-733.
[23] ZHANG BL, QIN DY, MO ZM, et al. Hurdles of CAR-T cell-based cancer immunotherapy directed against solid tumors. Sci China Life Sci,2016,59(4):340-348.
[24] ZHANG H, YE ZL, YUAN ZG, et al. New Strategies for the treatment of solid tumors with CAR-T cells. Int J Biol Sci,2016,12(6):718-729.
[25] PARK HJ, KUSNADI A, LEE EJ, et al. Tumor-infiltrating regulatory T cells delineated by upregulation of PD-1 and inhibitory receptors. Cell Immunol,2012,278(1/2):76-83.
[26] BROWN CE, ALIZADEH D, STARR R, et al. Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J Med,2016,375(26):2561-2569.
[27] KATZ SC, BURGA RA, MCCORMACK E, et al. Phase I hepatic immunotherapy for metastases study of intra-arterial chimeric antigen receptor-modified T-cell therapy for CEA+ liver metastases. Clin Cancer Res,2015,21(14):3149-3159.
[28] ZHANG H, QIU J, YE C, et al. ROR1 expression correlated with poor clinical outcome in human ovarian cancer. Sci Rep,2014,4:5811[2018-09-16].https://www.nature.com/articles/srep05811. doi:10.1038/srep05811.