|Table of Contents|

Construction of T cells expressing chimeric antigen receptor targeting prostate stem cell antigen and its anti-tumor effect in tumor-bearing mice



Research Field:
Publishing date:



Construction of T cells expressing chimeric antigen receptor targeting prostate stem cell antigen and its anti-tumor effect in tumor-bearing mice


HONG Juan CHEN Yunfan SHEN Junjie XU Yanmin QIAN Cheng

Biological Therapy Center, First Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400038, China


chimeric antigen receptor prostate stem cell antigen tumor immunotherapy lentivirus


ObjectiveTo construct genetically modified T cells expressing chimeric antigen receptor (CAR) targeting prostate stem cell antigen (PSCA) and test the cytotoxicity of these cells against tumor cells both in vitro and in a tumorbearing mouse model. MethodsThe sequences encoding the light and heavy chain variable regions of a monoclonal antibody targeting PSCA were synthesized and integrated into lentiviral vectors using restriction enzymes. Human T lymphocytes isolated from peripheral blood mononuclear cells were infected with the recombinant lentivirus and the positivity rate of PSCACAR expression in the T lymphocytes was detected using flow cytometry. Cytotoxic assay was performed to evaluate the specific cytotoxicity of the genetically modified T cells against the PSCApositive HeLa cells and PSCAnegotive T24 cells, and ELISA was used to detect cytokine secretion by the cells. The antitumor effect of PSCACART cells was also tested in a NOG mouse model bearing HeLa cell xenograft. ResultsDNA sequencing analysis suggested correct synthesis of PSCACAR sequences, and enzyme digestion verified successful construction of the lentiviral vector PLPSCAG4H28TM28BBζ. The positivity rate of CAR expression was 53.9% in the T lymphocytes after infection with the lentiviral vector. The PSCACART cells showed a significantly stronger cytotoxicity against PSCApositive tumor cells than against PSCAnegative tumor cells (P<001) both in shortterm coincubation at a high effectivetarget ratio and in longterm coincubation at a low effectivetarget ratio. The PSCACART cells secreted significantly higher levels of cytokines than the control T cells upon stimulation by PSCApositive tumor cells (P<001), but the secretions were almost undetectable upon stimulation by PSCAnegative tumor cells. In the in vivo experiment, PSCACART cells exhibited a significant tumorsuppressive effect in the tumorbearing mice (P<001). ConclusionPSCACART cells capable of specific killing of PSCApositive tumor cells are successfully constructed both in vitro and in vivo.


[1]KOWOLIK C M, TOPP M S, GONZALEZ S, et al. CD28 costimulation provided through a CD19specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells[J]. Cancer Res, 2006,66(22): 10995-11004. DOI: 10.1158/00085472.CAN060160.
[2]SADELAIN M, BRENTJENS R, RIVIRE I. The basic principles of chimeric antigen receptor design[J]. Cancer Discov, 2013,3(4): 388-398. DOI: 10.1158/21598290.CD 120548.
[3]REITER R E, GU Z, WATABE T, et al. Prostate stem cell antigen:  a cell surface marker overexpressed in prostate cancer[J]. Proc Natl Acad Sci U S A, 1998, 95(4): 1735-1740.
[4]YANG X, GUO Z, LIU Y, et al. Prostate stem cell antigen and cancer risk, mechanisms and therapeutic implications[J]. Expert Rev Anticancer Ther, 2014, 14(1): 31-37. DOI:  10.1586/14737140.2014.845372.
[5]Study Group of Millennium Genome Project for Cancer, SAKAMOTO H, YOSHIMURA K, et al. Genetic variation in PSCA is associated with susceptibility to diffusetype gastric cancer[J]. Nat Genet, 2008, 40(6): 730-740. DOI:  10.1038/ng.152.
[6]LOCHHEAD P, FRANK B, HOLD G L, et al. Genetic variation in the prostate stem cell antigen gene and upper gastrointestinal cancer in white individuals[J]. Gastroenterology, 2011, 140(2): 435-441. DOI:  10.1053/j.gastro.2010.11.001.
[7]LU Y, CHEN J, DING Y, et al. Genetic variation of PSCA gene is associated with the risk of both diffuse and intestinaltype gastric cancer in a Chinese population[J]. Int J Cancer, 2010, 127(9): 2183-2189. DOI: 10.1002/ijc.25228.
[8]MATSUO K, TAJIMA K, SUZUKI T, et al. Association of prostate stem cell antigen gene polymorphisms with the risk of stomach cancer in Japanese[J]. Int J Cancer, 2009, 125(8): 1961-1964. DOI:  10.1002/ijc.24519.
[9]GRUBBS E G, ABDELWAHAB Z, TYLER D S, et al. Utilizing quantitative polymerase chain reaction to evaluate prostate stem cell antigen as a tumor marker in pancreatic cancer[J]. Ann Surg Oncol, 2006,13(12): 1645-1654. DOI:  10.1245/s1043400690295.
[10]WU X, YE Y, KIEMENEY L A, et al. Genetic variation in the prostate stem cell antigen gene PSCA confers susceptibility to urinary bladder cancer[J]. Nat Genet, 2009, 41(9): 991-995. DOI:  10.1038/ng.421.
[11]WANG S, TANG J, WANG M, et al. Genetic variation in PSCA and bladder cancer susceptibility in a Chinese population[J]. Carcinogenesis, 2010, 31(4): 621-624. DOI: 10.1093/carcin/bgp323.
[12]MARRA E, UVA P, VITI V, et al. Growth delay of human bladder cancer cells by prostate stem cell antigen downregulation is associated with activation of immune signaling pathways[J]. BMC Cancer, 2010, 10:129.DOI: 10.1186/1471240710129.
[13]DANNULL J, DIENER P A, PRIKLER L, et al. Prostate stem cell antigen is a promising candidate for immunotherapy of advanced prostate cancer[J]. Cancer Res, 2000, 60(19): 5522-5528.
[14]MORGENROTH A, CARTELLIERI M, SCHMITZ M, et al. Targeting of tumor cells expressing the prostate stem cell antigen (PSCA) using genetically engineered Tcells[J]. Prostate, 2007, 67(10): 1121-1131. DOI:  10.1002/pros.20608.
[15]JENA B, DOTTI G, COOPER L J. Redirecting Tcell specificity by introducing a tumorspecific chimeric antigen receptor[J]. Blood, 2010, 116(7): 1035-1044. DOI: 10.1182/blood201001043737.
[16]PORTER D L, LEVINE B L, KALOS M, et al. Chimeric antigen receptormodified T cells in chronic lymphoid leukemia[J]. N Engl J Med, 2011, 365(8): 725-733. DOI: 10.1056/NEJMoa1103849.
[17]GARFALL A L, MAUS M V, HWANG W T, et al. Chimeric antigen receptor T cells against CD19 for multiple myeloma[J]. N Engl J Med, 2015,373: 1040-1047. DOI: 10.1056/NEJMoa1504542.
[18]TASIAN S K, KENDERIAN S S, SHEN F, et al. Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia[J]. Blood, 2017,129(17): 2395-2407. DOI: 10.1182/blood201608736041.
[19]ORMH J M, BEDOYA F, FRIGAULT M J, et al. CARs in the lead against multiple myeloma[J]. Curr Hematol Malig Rep, 2017, 12(2): 119-125. DOI:  10.1007/s1189901703732.
[20]SAFFRAN D C, RAITANO A B, HUBERT R S, et al. AntiPSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts[J]. Proc Natl Acad Sci U S A, 2001, 98(5): 2658-2663. DOI:  10.1073/pnas.051624698.
[21]WEI X, LAI Y, LI J, et al. PSCA and MUC1 in nonsmallcell lung cancer as targets of chimeric antigen receptor T cells[J]. Oncoimmunology, 2017, 6(3): e1284722. DOI: 10.1080/2162402X.2017.1284722. 


Last Update: 2018-07-02