Tumor Biology

, Volume 37, Issue 1, pp 799–806 | Cite as

Engineered cytotoxic T lymphocytes with AFP-specific TCR gene for adoptive immunotherapy in hepatocellular carcinoma

  • Longhao Sun
  • Hao Guo
  • Ruoyu Jiang
  • Li Lu
  • Tong Liu
  • Xianghui HeEmail author
Original Article


Alpha-fetoprotein (AFP) is overexpressed in hepatocellular carcinoma (HCC) and could serve as a tumor-associated antigen (TAA) and potential target for adoptive immunotherapy. However, low frequency and severe functional impairment of AFP-specific T cells in vivo hamper adoptive infusion. TAA-specific T cell receptor (TCR) gene transfer could be an efficient and reliable alternation to generate AFP-specific cytotoxic T lymphocytes (CTLs). Autologous dendritic cells (DC) pulsed with AFP158-166 peptides were used to stimulate AFP-specific CTLs. TCR α/β chain genes of AFP-specific CTLs were cloned and linked by 2A peptide to form full-length TCR coding sequence synthesized into a lentiviral vector. Nonspecific activated T cells were engineered by lentivirus infection. Transgenetic CTLs were evaluated for transfection efficiency, expression of AFP158-166-specific TCR, interferon (IFN)-γ secretion, and specific cytotoxicity toward AFP+ HCC cells in vitro and in vivo. Flow cytometry revealed the AFP158-166-MHC-Pentamer positive transgenetic CTLs was 9.86 %. The number of IFN-γ secretion T cells and the specific cytotoxicity toward HpeG2 in vitro and in tumor-bearing NOD/SCID mice were significantly raised in transgenetic CTLs than that of AFP158-166-specific CTLs obtained by peptide-pulsed DCs or control group. TCR gene transfer is a promising strategy to generate AFP158-166-specific CTLs for the treatment of HCC.


AFP158-166-specific T cell receptor Transgenetic Adoptive immunotherapy Hepatocellular carcinoma 



This work was supported by National Natural Science Foundation of China Grants No.81172167.

Conflicts of interest



  1. 1.
    Mittal S, El-Serag HB. Epidemiology of hepatocellular carcinoma: consider the population. J Clin Gastroenterol. 2013;47(Suppl):S2–6.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Cucchetti A, Qiao GL, Cescon M, Li J, Xia Y, Ercolani G, et al. Anatomic versus nonanatomic resection in cirrhotic patients with early hepatocellular carcinoma. Surgery. 2014;155:512–21.CrossRefPubMedGoogle Scholar
  3. 3.
    Weiner LM, Dhodapkar MV, Ferrone S. Monoclonal antibodies for cancer immunotherapy. Lancet. 2009;373:1033–40.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Gehring AJ, Xue SA, Ho ZZ, Teoh D, Ruedl C, Chia A, et al. Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines. J Hepatol. 2011;55:103–10.CrossRefPubMedGoogle Scholar
  5. 5.
    Kalos M, June CH. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity. 2013;39:49–60.CrossRefPubMedGoogle Scholar
  6. 6.
    El-Serag HB, Kanwal F. α-Fetoprotein in hepatocellular carcinoma surveillance: mend it but do not end it. Clin Gastroenterol Hepatol. 2013;11:441–3.CrossRefPubMedGoogle Scholar
  7. 7.
    Butterfield LH, Ribas A, Meng WS, Dissette VB, Amarnani S, Vu HT, et al. T-cell responses to HLA-A*0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer. Clin Cancer Res. 2003;9:5902–8.PubMedGoogle Scholar
  8. 8.
    Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME, et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science. 2014;344:641–5.CrossRefPubMedGoogle Scholar
  9. 9.
    Visioni A, Zhang M, Graor H, Kim J. Expansion of melanoma-specific T cells from lymph nodes of patients in stage III: implications for adoptive immunotherapy in treating cancer. Surgery. 2012;152:557–65. discussion 565-6.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Bonaccorsi I, Pezzino G, Morandi B, Ferlazzo G. Novel perspectives on dendritic cell-based immunotherapy of cancer. Immunol Lett. 2013;155:6–10.CrossRefPubMedGoogle Scholar
  11. 11.
    Yang S, Dudley ME, Rosenberg SA, Morgan RA. A simplified method for the clinical-scale generation of central memory-like CD8+ T cells after transduction with lentiviral vectors encoding antitumor antigen T-cell receptors. J Immunother. 2010;33:648–58.CrossRefPubMedGoogle Scholar
  12. 12.
    Wu F, Zhang W, Shao H, Bo H, Shen H, Li J, et al. Human effector T cells derived from central memory cells rather than CD8(+)T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy. Cancer Lett. 2013;339:195–207.CrossRefPubMedGoogle Scholar
  13. 13.
    Perro M, Tsang J, Xue SA, Escors D, Cesco-Gaspere M, Pospori C, et al. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer. Gene Ther. 2010;17:721–32.CrossRefPubMedGoogle Scholar
  14. 14.
    Clay TM, Custer MC, Sachs J, Hwu P, Rosenberg SA, Nishimura MI. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers antitumor reactivity. J Immunol. 1999;163:507–13.PubMedGoogle Scholar
  15. 15.
    Frankel TL, Burns WR, Peng PD, Yu Z, Chinnasamy D, Wargo JA, et al. Both CD4 and CD8 T cells mediate equally effective in vivo tumor treatment when engineered with a highly avid TCR targeting tyrosinase. J Immunol. 2010;184:5988–98.CrossRefPubMedGoogle Scholar
  16. 16.
    Chinnasamy N, Wargo JA, Yu Z, Rao M, Frankel TL, Riley JP, et al. A TCR targeting the HLA-A*0201-restricted epitope of MAGE-A3 recognizes multiple epitopes of the MAGE-A antigen superfamily in several types of cancer. J Immunol. 2011;186:685–96.CrossRefPubMedGoogle Scholar
  17. 17.
    Straetemans T, van Brakel M, van Steenbergen S, Broertjes M, Drexhage J, Hegmans J, et al. TCR gene transfer: MAGE-C2/HLA-A2 and MAGE-A3/HLA-DP4 epitopes as melanoma-specific immune targets. Clin Dev Immunol. 2012;2012:586314.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Batchu RB, Gruzdyn OV, Moreno-Bost AM, Szmania S, Jayandharan G, Srivastava A, et al. Efficient lysis of epithelial ovarian cancer cells by MAGE-A3-induced cytotoxic T lymphocytes using rAAV-6 capsid mutant vector. Vaccine. 2014;32:938–43.CrossRefPubMedGoogle Scholar
  19. 19.
    Jakka G, Schuberth PC, Thiel M, Held G, Stenner F, Van Den Broek M, et al. Antigen-specific in vitro expansion of functional redirected NY-ESO-1-specific human CD8+ T-cells in a cell-free system. Anticancer Res. 2013;33:4189–201.PubMedGoogle Scholar
  20. 20.
    Schuberth PC, Jakka G, Jensen SM, Wadle A, Gautschi F, Haley D, et al. Effector memory and central memory NY-ESO-1-specific re-directed T cells for treatment of multiple myeloma. Gene Ther. 2013;20:386–95.CrossRefPubMedGoogle Scholar
  21. 21.
    Hillerdal V, Nilsson B, Carlsson B, Eriksson F, Essand M. T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2+ prostate and breast cancer cells. Proc Natl Acad Sci U S A. 2012;109:15877–81.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Butterfield LH, Ribas A, Dissette VB, Lee Y, Yang JQ, De la Rocha P, et al. A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four alpha-fetoprotein peptides. Clin Cancer Res. 2006;12:2817–25.CrossRefPubMedGoogle Scholar
  23. 23.
    Boria I, Cotella D, Dianzani I, Santoro C, Sblattero D. Primer sets for cloning the human repertoire of T cell receptor variable regions. BMC Immunol. 2008;9:50.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Fang J, Qian JJ, Yi S, Harding TC, Tu GH, VanRoey M, et al. Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol. 2005;23:584–90.CrossRefPubMedGoogle Scholar
  25. 25.
    Cohen CJ, Zhao Y, Zheng Z, Rosenberg SA, Morgan RA. Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability. Cancer Res. 2006;66:8878–86.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Cohen CJ, Li YF, El-Gamil M, Robbins PF, Rosenberg SA, Morgan RA. Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond. Cancer Res. 2007;67:3898–903.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Kuball J, Dossett ML, Wolfl M, Ho WY, Voss RH, Fowler C, et al. Facilitating matched pairing and expression of TCR chains introduced into human T cells. Blood. 2007;109:2331–8.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Voss RH, Willemsen RA, Kuball J, Grabowski M, Engel R, Intan RS, et al. Molecular design of the Calphabeta interface favors specific pairing of introduced TCRalphabeta in human T cells. J Immunol. 2008;180:391–401.CrossRefPubMedGoogle Scholar
  29. 29.
    Okamoto S, Mineno J, Ikeda H, Fujiwara H, Yasukawa M, Shiku H, et al. Improved expression and reactivity of transduced tumor-specific TCRs in human lymphocytes by specific silencing of endogenous TCR. Cancer Res. 2009;69:9003–11.CrossRefPubMedGoogle Scholar
  30. 30.
    Ochi T, Fujiwara H, Okamoto S, An J, Nagai K, Shirakata T, et al. Novel adoptive T-cell immunotherapy using a WT1-specific TCR vector encoding silencers for endogenous TCRs shows marked antileukemia reactivity and safety. Blood. 2011;118:1495–503.CrossRefPubMedGoogle Scholar
  31. 31.
    Provasi E, Genovese P, Lombardo A, Magnani Z, Liu PQ, Reik A, et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med. 2012;18:807–15.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Longhao Sun
    • 1
  • Hao Guo
    • 1
  • Ruoyu Jiang
    • 1
  • Li Lu
    • 1
  • Tong Liu
    • 1
  • Xianghui He
    • 1
    Email author
  1. 1.Department of General SurgeryTianjin Medical University General HospitalTianjinChina

Personalised recommendations