The Construction of ROR1 Targeting Chimeric Antigen Receptor Modified T Cells and Its Killing Effect for ROR1-positive Tumor Cells

CHEN Yue, MO Ze-ming, QIN Di-yuan.et al

Abstract

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. MethodsThe 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. ResultsThe 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). ConclusionWe 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.

 

Keywords: CAR-TROR1Immunotherapy 

 

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References


WILSON C, GOBERDHAN ГХ'. STELLER H. Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family of Trk-related receptor tyrosine kinases. Proc Natl Acad Sci U S A. 1993. 90( 15): 7109-7113.

SHABANI M. ASG ARIAN-OMR A N H, VOSSOUGH I, et al. Expression profile of orphan receptor tyrosine kinase (ROR1) and Wilms’ tumor gene 1 (WT1 ) in different subsets of В-cell acute lymphoblastic leukemia. Leuk ymphoma,2008,49(7): 1360-1367.

BALAKRISHNAN A, GOODPASTER T. RANDOLPH- HABECKER J. et al. Analysis of RORl protein expression in human cancer and normal tissues. Clin Cancer Res. 2016. 23(12);3061-3071.

BASKAR S, KWONG KY, HOFER T, et al. Unique cell surface expression of receptor tyrosine kinase RORl in human В-cell chronic lymphocytic leukemia. Clin Cancer Res, 2008, 14(2);396-404.

DANESHMANESH AH. PORWIT A, HOJJAT-FARSANGI M, et al. Orphan receptor tyrosine kinases RORl and ROR2 in hematological malignancies. Leuk Lymphoma, 2013,54(4): 843-850.

RABBANI H, OSTADKARAMPOUR M. DANESH MANESH AH. et al. Expression of RORl in patients with renal cancer—a potential diagnostic marker. Iran Biomed J, 2010.14(3):77-82.

ZHANGS, CHEN L. WANG-RODRIGUEZ J, et al. The onco-embryonic antigen RORl is expressed by a variety of human cancers. Am J Pathol.2012.181(6); 1903-1910.

ZHANG S, CHEN L, CUI B, et al. RORl 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.

CUI B, ZHANG S, CHEN L, et al. Targeting RORl inhibits epithelial-mesenchymal transition and metastasis. Cancer Res, 2013,73(12): 3649-3660.

HOJJAT-FARSANGI M, GHAEMIMANESH F, DANESHMANESH AH. et al. Inhibition of the receptor tyrosine kinase RORl by anti-RORl 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.

DANESHMANESH AH. HOJ J AT-FARSANGI M, KHAN AS, et al. Monoclonal antibodies against RORl induce apoptosis of chronic lymphocytic leukemia ( CLL) cells. Leukemia, 2012,26(6): 1348-1355.

SADELAIN M. BRENTJENS R. RIVIERE I. The promise and potential pitfalls of chimeric antigen receptors. Curr Opin Immunol, 2009,21 (2): 215-223.

HOMBACH A, HOMBACH AA, ABKEN H. Adoptive immunotherapy with genetically engineered T cells; modification of the IgGl Fc 4 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.

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.

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.

S ADEL AIN M, BRENT JENS R. RIVIERE I. The basic principles of chimeric antigen receptor design. Cancer Discov, 2013,3(4):388-398.

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.

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.

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.

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.

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 В-cell leukemias. Blood, 2011, 118 ( 18 ): 4817- 4828.

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.

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.

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.

PARK HJ, KUSNADI A, LEEEJ, et al. Tumor-infiltrating regulatory T cells delineated by upregulation of PI>1 and inhibitory receptors. Cell Immunol,2012,278( 1/2) :76-83.

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.

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.

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.


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