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Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma

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Abstract

Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1+GPC3− tumor cells, but they exhibited a similar cytotoxicity against GPC3+ASGR1− and GPC3+ASGR1+ HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3+ASGR1+ HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3+ASGR1+ HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3+ASGR1+ HCC.

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Abbreviations

ASGR1:

Asialoglycoprotein receptor 1

Bcl-xL:

B cell lymphoma-extra large

CAR-T:

Cells chimeric antigen receptor-modified T cells

eGFP:

Enhanced green fluorescent protein

ERK:

Extracellular regulated kinase

GPC3:

Glypican-3

HCC:

Hepatocellular carcinoma

MOI:

Multiplicity of infection

p-ERK:

Phosphorylated ERK

rhIL-2:

Recombinant human interleukin 2

Tcm:

Central memory T cell

TMA:

Tissue microarray

References

  1. Li ZH, Chen C, Li KS, Jiang H, Song F, Gao HP, Pan XR, Shi BZ, Bi YY, Wang HM, Wang HY (2016) Development of T-cells carrying two complementary chimeric antigen receptors against GPC3 and ASGR1 for the treatment of hepatocellular carcinoma. Mol Ther 24(supplement 1):S79. doi:10.1038/mt.2016.78

    Article  Google Scholar 

  2. Cancer IAfRo (2014) World cancer report 2014. WHO, Geneva

    Google Scholar 

  3. Llovet JM, Real MI, Montana X, Planas R, Coll S, Aponte J, Ayuso C, Sala M, Muchart J, Sola R, Rodes J, Bruix J, Barcelona Liver Cancer G (2002) Arterial embolisation or chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma: a randomised controlled trial. Lancet 359(9319):1734–1739. doi:10.1016/S0140-6736(02)08649-X

    Article  PubMed  Google Scholar 

  4. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Haussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J, Group SIS (2008) Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359(4):378–390. doi:10.1056/NEJMoa0708857

    Article  CAS  PubMed  Google Scholar 

  5. Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar Z, Mavroukakis SA, White DE, Wunderlich JR, Canevari S, Rogers-Freezer L, Chen CC, Yang JC, Rosenberg SA, Hwu P (2006) A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res 12(20 Pt 1):6106–6115. doi:10.1158/1078-0432.CCR-06-1183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Park JR, Digiusto DL, Slovak M, Wright C, Naranjo A, Wagner J, Meechoovet HB, Bautista C, Chang WC, Ostberg JR, Jensen MC (2007) Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma. Mol Ther 15(4):825–833. doi:10.1038/sj.mt.6300104

    Article  CAS  PubMed  Google Scholar 

  7. Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal M, Suhoski MM, Varela-Rohena A, Haines KM, Heitjan DF, Albelda SM, Carroll RG, Riley JL, Pastan I, June CH (2009) Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci USA 106(9):3360–3365. doi:10.1073/pnas.0813101106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, June CH (2011) T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 3(95):95ra73. doi:10.1126/scitranslmed.3002842

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Porter DL, Levine BL, Kalos M, Bagg A, June CH (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365(8):725–733. doi:10.1056/NEJMoa1103849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gao H, Li K, Tu H, Pan X, Jiang H, Shi B, Kong J, Wang H, Yang S, Gu J, Li Z (2014) Development of T cells redirected to glypican-3 for the treatment of hepatocellular carcinoma. Clin Cancer Res 20(24):6418–6428. doi:10.1158/1078-0432.CCR-14-1170

    Article  CAS  PubMed  Google Scholar 

  11. Baumhoer D, Tornillo L, Stadlmann S, Roncalli M, Diamantis EK, Terracciano LM (2008) Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples. Am J Clin Pathol 129(6):899–906. doi:10.1309/HCQWPWD50XHD2DW6

    Article  PubMed  Google Scholar 

  12. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA (2010) Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18(4):843–851. doi:10.1038/mt.2010.24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wilkie S, van Schalkwyk MC, Hobbs S, Davies DM, van der Stegen SJ, Pereira AC, Burbridge SE, Box C, Eccles SA, Maher J (2012) Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signaling. J Clin Immunol 32(5):1059–1070. doi:10.1007/s10875-012-9689-9

    Article  CAS  PubMed  Google Scholar 

  14. Kloss CC, Condomines M, Cartellieri M, Bachmann M, Sadelain M (2013) Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol 31(1):71–75. doi:10.1038/nbt.2459

    Article  CAS  PubMed  Google Scholar 

  15. Fiume L, Di Stefano G, Busi C, Mattioli A, Bonino F, Torrani-Cerenzia M, Verme G, Rapicetta M, Bertini M, Gervasi GB (1997) Liver targeting of antiviral nucleoside analogues through the asialoglycoprotein receptor. J Viral Hepat 4(6):363–370

    Article  CAS  PubMed  Google Scholar 

  16. Coulstock E, Sosabowski J, Ovecka M, Prince R, Goodall L, Mudd C, Sepp A, Davies M, Foster J, Burnet J, Dunlevy G, Walker A (2013) Liver-targeting of interferon-alpha with tissue-specific domain antibodies. PLoS ONE 8(2):e57263. doi:10.1371/journal.pone.0057263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mu H, Lin KX, Zhao H, **ng S, Li C, Liu F, Lu HZ, Zhang Z, Sun YL, Yan XY, Cai JQ, Zhao XH (2014) Identification of biomarkers for hepatocellular carcinoma by semiquantitative immunocytochemistry. World J Gastroenterol 20(19):5826–5838. doi:10.3748/wjg.v20.i19.5826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Witzigmann D, Quagliata L, Schenk SH, Quintavalle C, Terracciano LM, Huwyler J (2016) Variable asialoglycoprotein receptor 1 expression in liver disease: implications for therapeutic intervention. Hepatol Res 46(7):686–696. doi:10.1111/hepr.12599

    Article  CAS  PubMed  Google Scholar 

  19. Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DA (2004) Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 22(5):589–594. doi:10.1038/nbt957

    Article  CAS  PubMed  Google Scholar 

  20. Wang H, Zhou M, Shi B, Zhang Q, Jiang H, Sun Y, Liu J, Zhou K, Yao M, Gu J, Yang S, Mao Y, Li Z (2011) Identification of an exon 4-deletion variant of epidermal growth factor receptor with increased metastasis-promoting capacity. Neoplasia 13(5):461–471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kuroda H, Kutner RH, Bazan NG, Reiser J (2009) Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection. J Virol Methods 157(2):113–121. doi:10.1016/j.jviromet.2008.11.021

    Article  CAS  PubMed  Google Scholar 

  22. Li K, Pan X, Bi Y, Xu W, Chen C, Gao H, Shi B, Jiang H, Yang S, Jiang L, Li Z (2016) Adoptive immunotherapy using T lymphocytes redirected to glypican-3 for the treatment of lung squamous cell carcinoma. Oncotarget 7(3):2496–2507. doi:10.18632/oncotarget.6595

    PubMed  Google Scholar 

  23. Goldstein NI, Prewett M, Zuklys K, Rockwell P, Mendelsohn J (1995) Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin Cancer Res 1(11):1311–1318

    CAS  PubMed  Google Scholar 

  24. Shi B, Abrams M, Sepp-Lorenzino L (2013) Expression of asialoglycoprotein receptor 1 in human hepatocellular carcinoma. J Histochem Cytochem 61(12):901–909. doi:10.1369/0022155413503662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Di Tommaso L, Destro A, Seok JY, Balladore E, Terracciano L, Sangiovanni A, Iavarone M, Colombo M, Jang JJ, Yu E, ** SY, Morenghi E, Park YN, Roncalli M (2009) The application of markers (HSP70 GPC3 and GS) in liver biopsies is useful for detection of hepatocellular carcinoma. J Hepatol 50(4):746–754. doi:10.1016/j.jhep.2008.11.014

    Article  PubMed  Google Scholar 

  26. Li J, Gao JZ, Du JL, Wei LX (2014) Prognostic and clinicopathological significance of glypican-3 overexpression in hepatocellular carcinoma: a meta-analysis. World J Gastroenterol 20(20):6336–6344. doi:10.3748/wjg.v20.i20.6336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hass HG, Jobst J, Scheurlen M, Vogel U, Nehls O (2015) Gene expression analysis for evaluation of potential biomarkers in hepatocellular carcinoma. Anticancer Res 35(4):2021–2028

    CAS  PubMed  Google Scholar 

  28. Trere D, Fiume L, De Giorgi LB, Di Stefano G, Migaldi M, Derenzini M (1999) The asialoglycoprotein receptor in human hepatocellular carcinomas: its expression on proliferating cells. Br J Cancer 81(3):404–408. doi:10.1038/sj.bjc.6690708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Shindo G, Endo T, Onda M, Goto S, Miyamoto Y, Kaneko T (2013) Is the CD4/CD8 ratio an effective indicator for clinical estimation of adoptive immunotherapy for cancer treatment? JCT 4(8):1382–1390. doi:10.4236/jct.2013.48164

    Article  CAS  Google Scholar 

  30. Yang S, Gattinoni L, Liu F, Ji Y, Yu Z, Restifo NP, Rosenberg SA, Morgan RA (2011) In vitro generated anti-tumor T lymphocytes exhibit distinct subsets mimicking in vivo antigen-experienced cells. Cancer Immunol Immunother 60(5):739–749. doi:10.1007/s00262-011-0977-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sallusto F, Geginat J, Lanzavecchia A (2004) Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol 22:745–763. doi:10.1146/annurev.immunol.22.012703.104702

    Article  CAS  PubMed  Google Scholar 

  32. Croft M (2003) Costimulation of T cells by OX40, 4-1BB, and CD27. Cytokine Growth Factor Rev 14(3–4):265–273

    Article  CAS  PubMed  Google Scholar 

  33. Rudd CE, Schneider H (2003) Unifying concepts in CD28, ICOS and CTLA4 co-receptor signalling. Nat Rev Immunol 3(7):544–556. doi:10.1038/nri1131

    Article  CAS  PubMed  Google Scholar 

  34. Watts TH (2005) TNF/TNFR family members in costimulation of T cell responses. Annu Rev Immunol 23:23–68. doi:10.1146/annurev.immunol.23.021704.115839

    Article  CAS  PubMed  Google Scholar 

  35. Kohn DB, Dotti G, Brentjens R, Savoldo B, Jensen M, Cooper LJ, June CH, Rosenberg S, Sadelain M, Heslop HE (2011) CARs on track in the clinic. Mol Ther 19(3):432–438. doi:10.1038/mt.2011.1

    Article  CAS  PubMed Central  Google Scholar 

  36. Stauss HJ, Morris EC (2013) Immunotherapy with gene-modified T cells: limiting side effects provides new challenges. Gene Ther 20(11):1029–1032. doi:10.1038/gt.2013.34

    Article  CAS  PubMed  Google Scholar 

  37. Lanitis E, Poussin M, Klattenhoff AW, Song D, Sandaltzopoulos R, June CH, Powell DJ Jr (2013) Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo. Cancer Immunol Res 1(1):43–53. doi:10.1158/2326-6066.CIR-13-0008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Takezawa R, Shinzawa K, Watanabe Y, Akaike T (1993) Determination of mouse major asialoglycoprotein receptor cDNA sequence. Biochim Biophys Acta 1172(1):220–222

    Article  CAS  PubMed  Google Scholar 

  39. Soukharev S, Berlin WK, Hanover JA, Bethke B, Sauer B (2000) Organization of the mouse ASGR1 gene encoding the major subunit of the hepatic asialoglycoprotein receptor. Gene 241(2):233–240

    Article  CAS  PubMed  Google Scholar 

  40. Nakano K, Yoshino T, Nezu J, Tsunoda H, Igawa T, Konishi H, Tanaka M, Sugo I, Kawai S, Ishiguro T, Kinoshita Y (2007) Anti-glypican 3 antibody. US 2007/0190599 A l [United States Patent Application Publication]

  41. Weijtens ME, Hart EH, Bolhuis RL (2000) Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production. Gene Ther 7(1):35–42. doi:10.1038/sj.gt.3301051

    Article  CAS  PubMed  Google Scholar 

  42. Caruso HG, Hurton LV, Najjar A, Rushworth D, Ang S, Olivares S, Mi T, Switzer K, Singh H, Huls H, Lee DA, Heimberger AB, Champlin RE, Cooper LJ (2015) Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity. Cancer Res 75(17):3505–3518. doi:10.1158/0008-5472.CAN-15-0139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Long AH, Haso WM, Shern JF, Wanhainen KM, Murgai M, Ingaramo M, Smith JP, Walker AJ, Kohler ME, Venkateshwara VR, Kaplan RN, Patterson GH, Fry TJ, Orentas RJ, Mackall CL (2015) 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 21(6):581–590. doi:10.1038/nm.3838

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23(9):1126–1136. doi:10.1038/nbt1142

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The study was funded by the Supporting Programs of the National Natural Science Foundation of China (No. 81502672), the “13th Five-Year Plan” for Science and Technology Research of China (2016ZX10002014-009) and the One Hundred Talents Scientific Research Projects of Health System in Shanghai (XBR2013123).

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Authors and Affiliations

  1. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, No. 25/Ln2200, ** Gao, **aorong Pan, Bizhi Shi, Yanyu Bi, Hongyang Wang & Zonghai Li

  2. CARsgen Therapeutics, Shanghai, China

    Huamao Wang

  3. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, 225 Changhai Road, Shanghai, China

    Hongyang Wang

  4. National Center for Liver Cancer, Shanghai, China

    Hongyang Wang

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  1. Cheng Chen
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  2. Kesang Li
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  3. Hua Jiang
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Chen, C., Li, K., Jiang, H. et al. Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma. Cancer Immunol Immunother 66, 475–489 (2017). https://doi.org/10.1007/s00262-016-1949-8

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