Construction of chimeric antigen receptor‑modified T cells targeting EpCAM and assessment of their anti‑tumor effect on cancer cells

Zhou,Yan; Wen,Ping; Li,Mingmei; Li,Yaqi; Li,Xiao‑An

doi:10.3892/mmr.2019.10460

Construction of chimeric antigen receptor‑modified T cells targeting EpCAM and assessment of their anti‑tumor effect on cancer cells

Authors:
- Yan Zhou
- Ping Wen
- Mingmei Li
- Yaqi Li
- Xiao‑An Li
View Affiliations

Affiliations: Gastroenterology Tumor and Microenvironment Laboratory, Department of Gastroenterology, The First Affiliated Hospital of Chengdu Medical College, Chengdu Medical College, Chengdu, Sichuan 610041, P.R. China
Published online on: July 3, 2019 https://doi.org/10.3892/mmr.2019.10460
Pages: 2355-2364

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Colon cancer is a common malignancy worldwide and there is an urgent requirement to develop effective treatment strategies. In recent years, tumor immunotherapy has become a new method of effectively treating tumors. Chimeric antigen receptor (CAR) T cell technology combines the precise targeting specificity of monoclonal antibodies with the strong toxicity and persistence of cytotoxic T cells to specifically recognize tumor‑associated antigens and promote tumor cell death efficiently and permanently, without depending on major histocompatibility complex restriction. In the present study, epithelial cell adhesion molecule (EpCAM)‑targeting CAR T cells (EpCAM‑CAR‑T) were developed, and their ability to kill cancer cells in vitro was assessed. Firstly, an EpCAM‑CAR plasmid was constructed using molecular biology techniques, and transfected into T cells to obtain EpCAM‑CAR‑T cells. Transfection efficiency was assessed using reverse transcription‑quantitative PCR and flow cytometry. Next, the expression levels of EpCAM in five colon cancer cell lines were examined by western blotting and flow cytometry. Finally, the effect of EpCAM‑CAR‑T cells on cancer cell death was examined in vitro via co‑culture experiments. T cells stably expressing EpCAM‑CAR were successfully obtained, and the transduction efficiency according to flow cytometry was 50.4%. In vitro experiments showed that EpCAM‑CAR‑T cells exhibited a significantly higher apoptotic effect on cancer cells compared with untransfected T cells. Analyses also demonstrated that this effect was dependent on the ratio of EpCAM‑CAR‑T cells to tumor cells, and the expression of surface EpCAM. Similarly, the ELISA results showed that interleukin (IL)‑2 IL‑6 and interferon‑γ levels were significantly elevated following exposure to EpCAM‑CAR‑T cells compared to exposure to untransfected T cells, and were dependent on the number of EpCAM‑CAR‑T cells and the amount of EpCAM expressed on the surface of tumor cells. The present study provided a basis for the clinical application of CAR‑T cell therapy against solid tumors, and a provided a new strategy for the treatment of colon cancer.

View Figures

View References

1	Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D and Bray F: Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 136:E359–E386. 2015. View Article : Google Scholar : PubMed/NCBI
2	Jemal A, Siegel R, Ward E, Hao Y, Xu J and Thun MJ: Cancer statistics, 2009. CA Cancer J Clin. 59:225–249. 2009. View Article : Google Scholar : PubMed/NCBI
3	Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester RGS, Barzi A and Jemal A: Colorectal cancer statistics, 2017. CA Cancer J Clin. 67:177–193. 2017. View Article : Google Scholar : PubMed/NCBI
4	Pang Y, Hou X, Yang C, Liu Y and Jiang G: Advances on chimeric antigen receptor-modified T-cell therapy for oncotherapy. Mol Cancer. 17:912018. View Article : Google Scholar : PubMed/NCBI
5	Couzin-Frankel J: Breakthrough of the year 2013. Cancer immunotherapy. Science. 342:1432–1433. 2013. View Article : Google Scholar : PubMed/NCBI
6	Mellman I, Coukos G and Dranoff G: Cancer immunotherapy comes of age. Nature. 480:480–490. 2011. View Article : Google Scholar : PubMed/NCBI
7	Rosenberg SA and Restifo NP: Adoptive cell transfer as personalized immunotherapy for human cancer. Science. 348:62–68. 2015. View Article : Google Scholar : PubMed/NCBI
8	Tran E, Robbins PF and Rosenberg SA: ‘Final common pathway’ of human cancer immunotherapy: Targeting random somatic mutations. Nat Immunol. 18:255–262. 2017. View Article : Google Scholar : PubMed/NCBI
9	Crompton JG, Klemen N and Kammula US: Metastasectomy for tumor-infiltrating lymphocytes: An emerging operative indication in surgical oncology. Ann Surg Oncol. 25:565–572. 2018. View Article : Google Scholar : PubMed/NCBI
10	Kunert A, Obenaus M, Lamers CHJ, Blankenstein T and Debets R: T-cell receptors for clinical therapy: In vitro assessment of toxicity risk. Clin Cancer Res. 23:6012–6020. 2017. View Article : Google Scholar : PubMed/NCBI
11	Hay KA and Turtle CJ: Chimeric antigen receptor (CAR) T cells: Lessons learned from targeting of CD19 in B-cell malignancies. Drugs. 77:237–245. 2017. View Article : Google Scholar : PubMed/NCBI
12	Dominguez G: The CART gene: Structure and regulation. Peptides. 27:1913–1918. 2006. View Article : Google Scholar : PubMed/NCBI
13	Zhang C, Liu J, Zhong JF and Zhang X: Engineering CAR-T cells. Biomark Res. 5:222017. View Article : Google Scholar : PubMed/NCBI
14	Martowicz A, Seeber A and Untergasser G: The role of EpCAM in physiology and pathology of the epithelium. Histol Histopathol. 31:349–355. 2016.PubMed/NCBI
15	Jin Z, Maiti S, Huls H, Singh H, Olivares S, Mátés L, Izsvák Z, Ivics Z, Lee DA, Champlin RE and Cooper LJ: The hyperactive sleeping beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor. Gene Therapy. 18:849–856. 2011. View Article : Google Scholar : PubMed/NCBI
16	Hao H, Zhen Y, Wang Z, Chen F and Xie X: A novel therapeutic drug for colon cancer: EpCAM scFv-truncated protamine (tp)-siRNA. Cell Biol Int. 37:860–864. 2013. View Article : Google Scholar : PubMed/NCBI
17	Mala J, Puthong S, Maekawa H, Kaneko Y, Palaga T, Komolpis K and Sooksai S: Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155). J Genet Eng Biotechnol. 15:69–76. 2017. View Article : Google Scholar : PubMed/NCBI
18	Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal M, Suhoski MM, Varela-Rohena A, Haines KM, Heitjan DF, Albelda SM, et al: Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci USA. 106:3360–3365. 2009. View Article : Google Scholar : PubMed/NCBI
19	Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ, Carrum G, Kamble RT, Bollard CM, Gee AP, Mei Z, et al: CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest. 121:1822–1826. 2011. View Article : Google Scholar : PubMed/NCBI
20	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
21	Ang WX, Li Z, Chi Z, Du SH, Chen C, Tay JC, Toh HC, Connolly JE, Xu XH and Wang S: Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis. Oncotarget. 8:13545–13559. 2017. View Article : Google Scholar : PubMed/NCBI
22	Fisher DT, Appenheimer MM and Evans SS: The two faces of IL-6 in the tumor microenvironment. Semin Immunol. 26:38–47. 2014. View Article : Google Scholar : PubMed/NCBI
23	Nakajima C, Uekusa Y, Iwasaki M, Yamaguchi N, Mukai T, Gao P, Tomura M, Ono S, Tsujimura T, Fujiwara H and Hamaoka T: A role of interferon-gamma (IFN-gamma) in tumor immunity: T cells with the capacity to reject tumor cells are generated but fail to migrate to tumor sites in IFN-gamma-deficient mice. Cancer Res. 61:3399–3405. 2001.PubMed/NCBI
24	Rosenberg SA: IL-2: The first effective immunotherapy for human cancer. J Immunol. 192:5451–5458. 2014. View Article : Google Scholar : PubMed/NCBI
25	Lichty BD, Breitbach CJ, Stojdl DF and Bell JC: Going viral with cancer immunotherapy. Nat Rev Cancer. 14:559–567. 2014. View Article : Google Scholar : PubMed/NCBI
26	Rodgers DT, Mazagova M, Hampton EN, Cao Y, Ramadoss NS, Hardy IR, Schulman A, Du J, Wang F, Singer O, et al: Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies. Proc Natl Acad Sci USA. 113:E459–E468. 2016. View Article : Google Scholar : PubMed/NCBI
27	Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, Teachey DT, Chew A, Hauck B, Wright JF, et al: Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 368:1509–1518. 2013. View Article : Google Scholar : PubMed/NCBI
28	Porter DL, Levine BL, Kalos M, Bagg A and June CH: Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 365:725–733. 2011. View Article : Google Scholar : PubMed/NCBI
29	Schuster SJ, Svoboda J, Chong EA, Nasta SD, Mato AR, Anak Ö, Brogdon JL, Pruteanu-Malinici I, Bhoj V, Landsburg D, et al: Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med. 377:2545–2554. 2017. View Article : Google Scholar : PubMed/NCBI
30	Singh N, Shi J, June CH and Ruella M: Genome-editing technologies in adoptive T cell immunotherapy for cancer. Curr Hematol Malig Rep. 12:522–529. 2017. View Article : Google Scholar : PubMed/NCBI
31	Schmelzer E and Reid LM: EpCAM expression in normal, non-pathological tissues. Front Biosci. 13:3096–3100. 2008. View Article : Google Scholar : PubMed/NCBI
32	Dai M, Yuan F, Fu C and Shen G, Hu S and Shen G: Relationship between epithelial cell adhesion molecule (EpCAM) overexpression and gastric cancer patients: A systematic review and meta-analysis. PLoS One. 12:e01753572017. View Article : Google Scholar : PubMed/NCBI
33	Fong D, Moser P, Kasal A, Seeber A, Gastl G, Martowicz A, Wurm M, Mian C, Obrist P, Mazzoleni G and Spizzo G: Loss of membranous expression of the intracellular domain of EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer. Histopathology. 64:683–692. 2014. View Article : Google Scholar : PubMed/NCBI
34	Seeber A, Untergasser G, Spizzo G, Terracciano L, Lugli A, Kasal A, Kocher F, Steiner N, Mazzoleni G, Gastl G and Fong D: Predominant expression of truncated EpCAM is associated with a more aggressive phenotype and predicts poor overall survival in colorectal cancer. Int J Cancer. 139:657–663. 2016. View Article : Google Scholar : PubMed/NCBI
35	Yahyazadeh Mashhadi SM, Kazemimanesh M, Arashkia A, Azadmanesh K, Meshkat Z, Golichenari B and Sahebkar A: Shedding light on the EpCAM: An overview. J Cell Physiol. 234:12569–12580. 2019. View Article : Google Scholar : PubMed/NCBI
36	Cooper LJ, Topp MS, Serrano LM, Gonzalez S, Chang WC, Naranjo A, Wright C, Popplewell L, Raubitschek A, Forman SJ and Jensen MC: T-cell clones can be rendered specific for CD19: Toward the selective augmentation of the graft-versus-B-lineage leukemia effect. Blood. 101:1637–1644. 2003. View Article : Google Scholar : PubMed/NCBI
37	Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K, Chung SS, Stefanski J, Borquez-Ojeda O, Olszewska M, et al: Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med. 6:224ra252014. View Article : Google Scholar : PubMed/NCBI
38	Wang Y, Zhang WY, Han QW, Liu Y, Dai HR, Guo YL, Bo J, Fan H, Zhang Y, Zhang YJ, et al: Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma treated by CD20-directed chimeric antigen receptor-modified T cells. Clin Immunol. 155:160–175. 2014. View Article : Google Scholar : PubMed/NCBI
39	Till BG, Jensen MC, Wang J, Qian X, Gopal AK, Maloney DG, Lindgren CG, Lin Y, Pagel JM, Budde LE, et al: CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: Pilot clinical trial results. Blood. 119:3940–3950. 2012. View Article : Google Scholar : PubMed/NCBI
40	Dholaria BR, Bachmeier CA and Locke F: Mechanisms and management of chimeric antigen receptor T-cell therapy-related toxicities. BioDrugs. 33:45–60. 2019. View Article : Google Scholar : PubMed/NCBI
41	Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M, Grupp SA and Mackall CL: Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2:188–195. 2014. View Article : Google Scholar
42	Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Fry TJ, Orentas R, Sabatino M, Shah NN, et al: T-cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: A phase 1 dose-escalation trial. Lancet. 385:517–528. 2015. View Article : Google Scholar : PubMed/NCBI
43	Oluwole OO and Davila ML: At the bedside: Clinical review of chimeric antigen receptor (CAR) T cell therapy for B cell malignancies. J Leukoc Biol. 100:1265–1272. 2016. View Article : Google Scholar : PubMed/NCBI