Abstract
There have been several studies suggesting that cancer stem cells (CSCs) contribute to the high rates of recurrence and resistance to therapies observed in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) has been demonstrated to be a biomarker of CSCs and a potential therapeutic target in HCC. Here, we prepared two anti-EpCAM monoclonal antibodies (1H8 and 2F2) and an anti-EpCAM bispecific T cell engager (BiTE) 1H8/CD3, which was derived from 1H8, and used them to treat HCC in vitro and in vivo. The results demonstrated that all of the developed anti-EpCAM antibodies specifically bound to EpCAM. Neither anti-EpCAM monoclonal antibody had obvious anti-HCC activities in vitro or in vivo. However, anti-EpCAM BiTE 1H8/CD3 induced strong peripheral blood mononuclear cell-dependent cellular cytotoxicity in Huh-7 and Hep3B cells but not EpCAM-negative SK-Hep-1 cells. Notably, 1H8/CD3 completely inhibited the growth of Huh-7 and Hep3B xenografts in vivo. Treatment of the Huh-7 HCC xenografts with 1H8/CD3 significantly suppressed tumor proliferation and reduced the expression of most CSC biomarkers. Intriguingly, galectin-1 (Gal-1) overexpression inhibited 1H8/CD3-induced lymphocytotoxicity in HCCs while knockdown of Gal-1 increased the lymphocytotoxicity. Collectively, these results indicate that anti-EpCAM BiTE 1H8/CD3 is a promising therapeutic agent for HCC treatment. Gal-1 may contribute to the resistance of HCC cells to 1H8/CD3-induced lysis.
Similar content being viewed by others
References
Llovet JM, Burroughs A, Bruix J (2003) Hepatocellular carcinoma. Lancet 362:1907–1917
Carr BI (2004) Hepatocellular carcinoma: current management and future trends. Gastroenterology 127:S218–S224
Zhu AX (2006) Systemic therapy of advanced hepatocellular carcinoma: how hopeful should we be? Oncologist 11:790–800
Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY (2008) CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene 27:1749–1758
Martin-Padura I, Marighetti P, Agliano A, Colombo F, Larzabal L, Redrado M, Bleau AM, Prior C, Bertolini F, Calvo A (2012) Residual dormant cancer stem-cell foci are responsible for tumor relapse after antiangiogenic metronomic therapy in hepatocellular carcinoma xenografts. Lab Investig 92:952–966
Xu XL, Xing BC, Han HB, Zhao W, Hu MH, Xu ZL, Li JY, Xie Y, Gu J, Wang Y, Zhang ZQ (2010) The properties of tumor-initiating cells from a hepatocellular carcinoma patient’s primary and recurrent tumor. Carcinogenesis 31:167–174
Sun YF, Xu Y, Yang XR, Guo W, Zhang X, Qiu SJ, Shi RY, Hu B, Zhou J, Fan J (2013) Circulating stem cell-like epithelial cell adhesion molecule-positive tumor cells indicate poor prognosis of hepatocellular carcinoma after curative resection. Hepatology 57:1458–1468
Coghlin C, Murray GI (2010) Current and emerging concepts in tumour metastasis. J Pathol 222:1–15
Yao Z, Mishra L (2009) Cancer stem cells and hepatocellular carcinoma. Cancer Biol Ther 8:1691–1698
Klonisch T, Wiechec E, Hombach-Klonisch S, Ande SR, Wesselborg S, Schulze-Osthoff K, Los M (2008) Cancer stem cell markers in common cancers—therapeutic implications. Trends Mol Med 14:450–460
Dingli D, Michor F (2006) Successful therapy must eradicate cancer stem cells. Stem Cells 24:2603–2610
Balzar M, Briaire-de Bruijn IH, Rees-Bakker HA, Prins FA, Helfrich W, de Leij L, Riethmuller G, Alberti S, Warnaar SO, Fleuren GJ, Litvinov SV (2001) Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions. Mol Cell Biol 21:2570–2580
Baeuerle PA, Gires O (2007) EpCAM (CD326) finding its role in cancer. Br J Cancer 96:417–423
Litvinov SV, Velders MP, Bakker HA, Fleuren GJ, Warnaar SO (1994) Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule. J Cell Biol 125:437–446
Maetzel D, Denzel S, Mack B, Canis M, Went P, Benk M, Kieu C, Papior P, Baeuerle PA, Munz M, Gires O (2009) Nuclear signalling by tumour-associated antigen EpCAM. Nat Cell Biol 11:162–171
Munz M, Kieu C, Mack B, Schmitt B, Zeidler R, Gires O (2004) The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene 23:5748–5758
Trzpis M, McLaughlin PM, de Leij LM, Harmsen MC (2007) Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule. Am J Pathol 171:386–395
Yamashita T, Budhu A, Forgues M, Wang XW (2007) Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma. Cancer Res 67:10831–10839
Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, Jia H, Ye Q, Qin LX, Wauthier E, Reid LM, Minato H, Honda M, Kaneko S, Tang ZY, Wang XW (2009) EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology 136:1012–1024
Yamashita T, Forgues M, Wang W, Kim JW, Ye Q, Jia H, Budhu A, Zanetti KA, Chen Y, Qin LX, Tang ZY, Wang XW (2008) EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res 68:1451–1461
Bae JS, Noh SJ, Jang KY, Park HS, Chung MJ, Park CK, Moon WS (2012) Expression and role of epithelial cell adhesion molecule in dysplastic nodule and hepatocellular carcinoma. Int J Oncol 41:2150–2158
McLaughlin PM, Harmsen MC, Dokter WH, Kroesen BJ, van der Molen H, Brinker MG, Hollema H, Ruiters MH, Buys CH, de Leij LF (2001) The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a new model to study carcinoma-directed immunotherapy. Cancer Res 61:4105–4111
Armstrong A, Eck SL (2003) EpCAM: a new therapeutic target for an old cancer antigen. Cancer Biol Ther 2:320–326
Chaudry MA, Sales K, Ruf P, Lindhofer H, Winslet MC (2007) EpCAM an immunotherapeutic target for gastrointestinal malignancy: current experience and future challenges. Br J Cancer 96:1013–1019
Cioffi M, Dorado J, Baeuerle PA, Heeschen C (2012) EpCAM/CD3-bispecific T-cell engaging antibody MT110 eliminates primary human pancreatic cancer stem cells. Clin Cancer Res 18:465–474
Herrmann I, Baeuerle PA, Friedrich M, Murr A, Filusch S, Ruttinger D, Majdoub MW, Sharma S, Kufer P, Raum T, Munz M (2010) Highly efficient elimination of colorectal tumor-initiating cells by an EpCAM/CD3-bispecific antibody engaging human T cells. PLoS One 5:e13474
Haas C, Krinner E, Brischwein K, Hoffmann P, Lutterbuse R, Schlereth B, Kufer P, Baeuerle PA (2009) Mode of cytotoxic action of T cell-engaging BiTE antibody MT110. Immunobiology 214:441–453
Brischwein K, Schlereth B, Guller B, Steiger C, Wolf A, Lutterbuese R, Offner S, Locher M, Urbig T, Raum T, Kleindienst P, Wimberger P, Kimmig R, Fichtner I, Kufer P, Hofmeister R, da Silva AJ, Baeuerle PA (2006) MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors. Mol Immunol 43:1129–1143
Jiang H, Wang H, Tan Z, Hu S, Shi B, Yang L, Li P, Gu J, Li Z (2011) Growth suppression of human hepatocellular carcinoma xenografts by a monoclonal antibody CH12 directed to epidermal growth factor receptor variant III. J Biol Chem 286:5913–5920
Yang Y, Jiang H, Gao H, Kong J, Zhang P, Hu S, Shi B, Yao M, Li Z (2012) The monoclonal antibody CH12 enhances the sorafenib-mediated growth inhibition of hepatocellular carcinoma xenografts expressing epidermal growth factor receptor variant III. Neoplasia 14:509–518
Osta WA, Chen Y, Mikhitarian K, Mitas M, Salem M, Hannun YA, Cole DJ, Gillanders WE (2004) EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res 64:5818–5824
Munz M, Murr A, Kvesic M, Rau D, Mangold S, Pflanz S, Lumsden J, Volkland J, Fagerberg J, Riethmuller G, Ruttinger D, Kufer P, Baeuerle PA, Raum T (2010) Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies. Cancer Cell Int 10:44
Chen Y, Yu D, Zhang H, He H, Zhang C, Zhao W, Shao RG (2012) CD133(+)EpCAM(+) phenotype possesses more characteristics of tumor initiating cells in hepatocellular carcinoma Huh7 cells. Int J Biol Sci 8:992–1004
Azuma T, Yao S, Zhu G, Flies AS, Flies SJ, Chen L (2008) B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells. Blood 111:3635–3643
Rousalova I, Krepela E (2010) Granzyme B-induced apoptosis in cancer cells and its regulation (review). Int J Oncol 37:1361–1378
Medema JP, de Jong J, van Hall T, Melief CJ, Offringa R (1999) Immune escape of tumors in vivo by expression of cellular FLICE-inhibitory protein. J Exp Med 190:1033–1038
Cedeno-Laurent F, Dimitroff CJ (2012) Galectin-1 research in T cell immunity: past, present and future. Clin Immunol 142:107–116
Wimberger P, Gilet H, Gonschior AK, Heiss MM, Moehler M, Oskay-Oezcelik G, Al-Batran SE, Schmalfeldt B, Schmittel A, Schulze E, Parsons SL (2012) Deterioration in quality of life (QoL) in patients with malignant ascites: results from a phase II/III study comparing paracentesis plus catumaxomab with paracentesis alone. Ann Oncol 23:1979–1985
Baumann K, Pfisterer J, Wimberger P, Burchardi N, Kurzeder C, du Bois A, Loibl S, Sehouli J, Huober J, Schmalfeldt B, Vergote I, Luck HJ, Wagner U (2011) Intraperitoneal treatment with the trifunctional bispecific antibody Catumaxomab in patients with platinum-resistant epithelial ovarian cancer: a phase IIa study of the AGO Study Group. Gynecol Oncol 123:27–32
Schmidt M, Scheulen ME, Dittrich C, Obrist P, Marschner N, Dirix L, Ruttinger D, Schuler M, Reinhardt C, Awada A (2010) An open-label, randomized phase II study of adecatumumab, a fully human anti-EpCAM antibody, as monotherapy in patients with metastatic breast cancer. Ann Oncol 21:275–282
Strohlein MA, Lordick F, Ruttinger D, Grutzner KU, Schemanski OC, Jager M, Lindhofer H, Hennig M, Jauch KW, Peschel C, Heiss MM (2011) Immunotherapy of peritoneal carcinomatosis with the antibody catumaxomab in colon, gastric, or pancreatic cancer: an open-label, multicenter, phase I/II trial. Onkologie 34:101–108
Baeuerle PA, Reinhardt C (2009) Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res 69:4941–4944
Weiner LM, Murray JC, Shuptrine CW (2012) Antibody-based immunotherapy of cancer. Cell 148:1081–1084
Topp MS, Kufer P, Gokbuget N, Goebeler M, Klinger M, Neumann S, Horst HA, Raff T, Viardot A, Schmid M, Stelljes M, Schaich M, Degenhard E, Kohne-Volland R, Bruggemann M, Ottmann O, Pfeifer H, Burmeister T, Nagorsen D, Schmidt M, Lutterbuese R, Reinhardt C, Baeuerle PA, Kneba M, Einsele H, Riethmuller G, Hoelzer D, Zugmaier G, Bargou RC (2011) Targeted therapy with the T-cell-engaging antibody blinatumomab of chemotherapy-refractory minimal residual disease in B-lineage acute lymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival. J Clin Oncol 29:2493–2498
Espelt MV, Croci DO, Bacigalupo ML, Carabias P, Manzi M, Elola MT, Munoz MC, Dominici FP, Wolfenstein-Todel C, Rabinovich GA, Troncoso MF (2011) Novel roles of galectin-1 in hepatocellular carcinoma cell adhesion, polarization, and in vivo tumor growth. Hepatology 53:2097–2106
Ito K, Stannard K, Gabutero E, Clark AM, Neo SY, Onturk S, Blanchard H, Ralph SJ (2012) Galectin-1 as a potent target for cancer therapy: role in the tumor microenvironment. Cancer Metastasis Rev 31:763–778
Rubinstein N, Alvarez M, Zwirner NW, Toscano MA, Ilarregui JM, Bravo A, Mordoh J, Fainboim L, Podhajcer OL, Rabinovich GA (2004) Targeted inhibition of galectin-1 gene expression in tumor cells results in heightened T cell-mediated rejection: a potential mechanism of tumor-immune privilege. Cancer Cell 5:241–251
Acknowledgments
This study was supported by the Supporting Program of the “Twelfth Five-year Plan” for Science and Technology Research of China (Grant No. 2012ZX09103-301-005 and 2012ZX10002014-006), the National Natural Science Foundation of China (No. 81071746) and the National Basic Research Program (Grant No. 2010CB529902).
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Pengfei Zhang and Bizhi Shi have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhang, P., Shi, B., Gao, H. et al. An EpCAM/CD3 bispecific antibody efficiently eliminates hepatocellular carcinoma cells with limited galectin-1 expression. Cancer Immunol Immunother 63, 121–132 (2014). https://doi.org/10.1007/s00262-013-1497-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00262-013-1497-4